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PEAKS Studio Manual 5.0 - Bioinformatics Solutions Inc.

1. Matched peptides shown in blue SPIDER matches shown in red 1 MRPAVRALLA CAVLGLCLAD PERTVRWCTI STHEANKCAS FRENVLRILE an r SGPFVSCVKK TSHMDCIKAI SNNEADAVTL DGGLVYEAGL KPNNLKPVVA 101 EFHGTKDINPO THYYAVAVVE KDTDFKLNEL RGKKSCHTGL GRSAGWNIPM 151 AKLYKELPDP QESIQRAAAN FFSASCVPCA DOSSFPKLCQ LCAGKGTDKC 201 ACSNHEPYFG YSGAFKCLME GAGDVAFVKH STVFDNLPNP EDRKNYELLC 251 GDNTRESVDD YOECYLAMVP SHAVVARTVG GKEDVIWELL NHAQEHFGED 3001 KPDNFOQLFOS PHGKDLLFKD SADGFLKIPS KMDFELYLGY EYVTALONLR 351 ESKPPDSSKD ECMVKWCAIG HQERTKCDRW SGFSGGAIEC ETAENTEECI amp Accession ID Mass Display PEAKS Score 96 Coverage Query matched Marked Description S I DB Search F 5 li Q29443 TRFE_BOVIN 3 Serotransferrin Bos taurus z P00330 ADH1_YEAST 2 36691 957 1 59 37 2 31 1 Alcohol dehydrogenase 1 E Sequence Browser Sequence Comparison La 5 z NCBI BLAST search of Q29443 TRFE BOVI 2 Link to retrieve entries containing this sequence from NCBI Entrez E E Accession ID Description Q29443 TRFE BOVIN Serotransferrin Bos taurus Bovine E Peptides List ID Sequence PEAKS Score RSD M Z Z Mr Calc Delta Mass Error ppm File RT Scan Quality Peptides Spectrum 1 DNPQTHYYAVAVVK 99 0 0 0 802 91 2 1603 7993 0 0061 3 8056 OrbiSample new 0 072 0 788 HSTVFDNLPNPEDR H Spectrum 4 96 7 0 57 547 59 3 0 0 0 0107 6 5511 Orbisample new 1 347
2. Click OK This will launch PEAKS Protein ID and when completed click on the Protein View button The results should appear similar to those shown below gt 000 e ne ea Or Note that the top protein result is Human Filamin A with a score of 98 89 Ar 118 Accession ID Mass Display PEAKS Score Coverage ane Marked Description o DB Search P c L sp Pz1333 FLMA HLIMAM 1 2B0 36 56 Cid 34 59 253 6 F Filarnin 4 O5 Home sapiens GH F Hl s sp QaMzMi MYOF HLIMAM 2 23470647 53 35 1 721 2 Myorerlin O5 Hormea sapiens GN F z 5 sp Q s n1 CBIC SALTY 3 23034 43 E 34 43 524 d C Cobalt precorrin 8 methylmutase p HO sp PO4264 K2C1 HUMAN 5 66017 65 _ 1 33 33 407 3 O Keratin type II cytoskeletal 1 O52 sp A6VECS RPOB_ACTSZ 308 149313 25 eb 73 1 04 1 F DNA directed RMA polymerase sub 1 ed sp QS9seB5 svB RHILO 309 76610 53 22 24 1 53 1 Ghycyl hRAA synthetase beta subu do a sp P49951 CLH1 BOVIN 245 191587 25 20 59 0 54 1 Clathrin heavy chain 1 O5 Bos Eau c Ee s sp AsAeMS K2CI PANTR 5 65489 13 20 1 il 2 Keratin type II cytoskeletal 1 52 E splO75363 FLNB HUMAN E 278192 7 19 43 0 42 2 L Filamin B OS Home sapiens GN F j n sp Q z8s55 VES HP v amp 3 13 16317 283 T 15 61 4 25 1 Protein E6 O5 Human papillemavir i aorar man PES aca Rocmeshebu ogee bende OE dee 4 Running Quantification Select the PEAKS Protei
3. OQ48KS9 1A1I 79 36881 0 60 7 3 85 2 1 aminocycloprop Q6LNIO FTHS 81 61957 504 60 7 2 23 3 Fj Formate tetrahy lt Q3SZR3 A1A 85 23182 424 lu 50 7 5 45 1 Alpha 1 acid glyc amp Q6DKE1 CYC 88 11695 4607 E 60 7 9 52 1 O Cytochrome c te He PO0017 CYC 91 11682 467 m 60 7 9 52 1 Fj Cytochrome c A Q03131 ERY 124 365030 44 60 7 0 34 2 C Erythronolide syn H QI2BHE GLP 126 55435 76 l 60 7 2 01 1 Fj Glycerol kinase Modifications Sequence Browser Sequence Comparison NCBI BLAST search of POO 22 BGAL ECOL Link to retrieve entries containing this sequence From NCBI Entrez Accession ID Description P00722 BGAL ECOLI Beta galactosidase Escherichia coli strain K12 Peptides List ID Sequence PEAKS M Z Mr Calc Delta Error p File Scan Quality ScanM Frag i Peptides SpecOMMFMAVR 99 12 481 74 961 47296 0 0075 7 8082 TEST6 2 19 523 1056 0 773FT ICRJO FT ICRJO GE e Hit 1Y5QOQL 1506 6885 SpedTAAC 1 99 12 477 72 953 4277 0 0022 2 3046 TEST6 2 23 085 1189 0 755 FT ICRJO FT ICRJO SpecAPLDMDI 99 12 729 36 1456 7158 0 0104 7 1229 TEST6_2 23 954 1224 0 786 FT ICRJO FT ICRJO SpecHQQOFF 99 12 533 31 1264 6101 0 0046 3 6681 TEST6 2 27 1421356 0 784 FT ICR O FT ICRJO SpecGDFOFNISR 99 12 542 26 1082 5144 0 0089 8 232 TEST6 2 27 56 1374 0 781 FT ICRJO FT ICRJO H E Hit 6IGLNC 1
4. ID Sequence 43 Peptides un Spectrum 1 DMPOTHY YAVAVVK XTANDEM E value RSD Miz z Mr Calc Delta Mass Error ppm File RT Scan Q 3 8056 OrbiSample rmz 3 9455 OrbiSample mz 0 0061 ID Sequence OMSSA E value RSD Miz Z Mr Calc Delta Mass Error ppm File RT Scan Quality m gt ru iG Peptides E iD Spectrum 1 DNPQTHY YAVAVVK 1 17E 10 802 91 2 1603 7993 0 0061 3 8056lOrbisample mzX 0 072 0 788 0 0816 695 84 2 1389 6484 0 0171 12 2978 0rbisample mzX 0 31 4 0 785 6 75E 6 820 89 2 1639 759 0 0065 3 9455 0rbisample mzX 1 4218 0 787 The inChorus report contains most of the information that is seen in a PEAKS Protein ID results file page 55 Click on the Peptide View tab P id Spectrum 2 TSDANIN 4 WN Spectrum 5 HSTVFDNLPNPEDR ID Sequence Inchorus M Z Z Mr Calc Delta Error RT Qua PEAKS xXTANDEM OMSSA E id Peptides __ it __ o gt Spectrum 1 DNPQTHYYAVAVWK 99 98 802 91 2 1603 7993 0 0061 3 8056 0 07 0 788 99 0 1 9E 5 c Spectrum 3 ANELLINVK 97 38 507 3 1012 5916 0 0062 6 0879 0 36 0 781 97 38 i Spectrum 4 HSTVFDNLPNPEDR 1639 759 C TSDANIN 3 WNNLK 99 97 1389 6523 Spectrum 2 4 0 0 0132 9 4869 0 785 Spectrum 5 i 0 250 1132 152529 83 1 42 0 787 Spectrum 3 ANEILINVK 1012 5916 0 00
5. Instrument Default Input File Directory xj Search Engine _ 192 168 1 30 data LTQ FT RAW Juan Casado Ion Editor Browse Default Output Directory Output Directory Browse C derbyServer Project Folder Browse C derbyServer serverDB Temporary File Directory C temp_mgf Browse Default Configuration File Directory C Documents and Settings bsil peaks peaksconf xml Browse Default Log File Location C Documents and Settings bsil peaks peaks log Browse Apply Use the and boxes to expand and collapse the view General Preferences Default Input File Directory Select where your data is being inputted from using the Browse button Default Output File Directory PEAKS outputs your results to C derbyServer by default Select the Browse button to change this location Project folder PEAKS uses C derbyServer serverDB as the default project folder Select the Browse button to change this location Temporary File Directory PEAKS uses C temp_mef as the default project folder Select the Browse button to change this location Default Configuration File Directory Your configuration files for PEAKS can be found at C Documents and Settings bsi peaks peaksconf xml by default Select the Browse button to change this location Default Log File Location Your log file for PEAKS can be found at C Documents and Settings bsi peaks peaks l
6. Marne Mana mass Residue site 4 hydroxynon 156 115 CHE Homoserine 7 E Hamoserine lac Hydroxylation 15 9949 PEDNRY ICPL heavy 111 0416 K X amp N TCPL light 105 0335 K X amp N Indnacetic acid Ws Methyl ester 14 0156 DE x ac Methylation 14 0156 CKRHDENQ 018 label 2 0042 BT X gc J Variable Modification IPropionamide 4 Deamidation Trimethylatian H Oxidation M Myristoylation N acyl diglvceri IN isapropylcar N Succinimidyl Oxidation M 15 2949 Oxidation HW 238 2297 C Show unimod Mew PTIM Max variable PTM per peptide Filter Options Filter the spectra which satisfy the Following conditions For use in the PTM search De nove amino acid score greater than 0 5 recommended 0 5 Protein ID peptide score less than 0 65 recommended 0 65 Saving the parameters for future reference 1s achieved by clicking on the Save Parameter button For more information on setting up PTM Finder parameters see page 63 Click OK to commence analysis 23 After the PTM Finder search is complete the Peptide View window will appear Sequence PEAKS Score RSD Miz Mricalc Delta Ma Errorippm File Pa Oe z HSTVFDMLPNP 99 0 1639 759 e Spectrum 4 395 68 OS 54759 3 ooj ooo amp SsilOrSamplem 1 347 0 777 0 Spectrum 5 o oga szos 2 0 0 D 0065 3 9455 OrbiSamplem 1428 0 787 es Spectrum 1 DNPQTHYYAVA S9 0 0 802 9
7. C Feaks Studio 5 0thirdpartylicenze txt Module License status PEAKS Protein ID Mever Expires OPERKSQ jNeverEgites O S O m View end user license agreement P Warning This computer program is protected by copyright avv and international treaties Unauthorized reproduction or distribution of this program or any portion of it may result in severe civil and criminal penalties and will be prosecuted ta the License Wizard L maximum extent possible under the law Tech support 2 5 Set up PEAKS Preferences Before running your data you must set up search engine preferences For an explanation on how to do this see page 93 It is possible to run your data through PEAKS without changing the other preferences as they have default settings For more information on changing these default settings see page 81 2 6 Set up PEAKS Configuration Before running your data you must configure your databases For instructions on how to do this see page 99 It is possible to run your data through PEAKS without configuring any other parameters as they have default settings For more information on changing these default settings see page 97 11 3 Quick Walkthrough This section of the manual will walk you through most of the basic functionality of PEAKS 5 After completing this section you will see how easy it is to load view a data file perform data refinement perform de novo sequencing and database
8. Iodoacetic acid derivative C Mannosylation 162 0528 Deamidation 0 98 EE 3 E iable gt d Variable Modification 3 9888 l Oxidation M Mr _ Remove a Deamidation 783 1415 Max variable PTM per peptide Parameters can be saved for future reference by clicking on the Save Parameter button For more information on setting up SPIDER Search parameters see page 68 commence analysis After the SPIDER search has completed the Peptide View window _ will appear The format 1S identical to what was seen in the results of a Protein ID search Click OK to LEE LS dare i7 m Lo i Slee D wm mcecu s 4 28 Jan 08 14 47 rising rra mca m FEE Peden 242 m x Ferien p rs TALE fore d n aur g Mr mic Dai n a Err Pis ET an T di Facer toni d parece He FY N 3 ir 3 5941 lN M Ohie em mp uU 2 Teheran Schum o TOAMNA ms 1 a 2T o LWBAAE awr VS ee A 1 TEE SDAA HT WSA Ls 1 de LLL a M3 M ieu dM oh a D C POP wey ee BI 3 8 3 Pr nds LL Imine d LTHET a LE T NN 3 ers LEUR LE eee Sem miML MC rrt A une la ZR i AE DD H8 ee ch amp 7T j amp H TA i4y 6 3 md mud La L Ia Li a E Y tas TET EEFT 5 TH ian ES Si TEES p 1 m n 3x i nili mtm zim vt SO M m 0 pA m m 5T EET cis 315 22 D pea Hira Na int p
9. CHPEAKS databaselNCBInrlgi taxid prot dmp gg 000 prot dmp ge T Download taxdmp CAPEAKS database NCBI nritaxdmp zip p Browse 99 2 Fasta Format Database Select your database from this drop down menu or select Other if your database 1s not in the list or if you would like to submit your own database Database Details FASTA format database MCBI nr Basic Options LUniProtKB Swiss Prot Database name Download Database Path 3 In the basics option panel enter a name for your database and select You have choose to download the database From Download Database Ftp Ftp ncbi nih gov blast db FASTA nr gz The following window will Click YES to invoke your default FTP client software and download automaticlly appear It may Fails due to the setting conflict of default FTP client software Download options Click NO Eo copy this URL to system clipboard You can start your FTP client software manually After download the database you need do decompression if it s a compressed File And then put the database File in the directory that PEAKS can access 4 If you would like to invoke your default FTP client software and download automatically click Yes If you select No the following window will appear telling you that the URL will be copied to your system clipboard Click Ok 1 j The URL to download the database has been copied to system clipboard Please start your
10. Retention Time 0 035 TIC 1 79E Number of Peaks 222 Fragmentation Type Unavailable Number of Results 7 DENOVO 3 19 Jan 09 17 28 has 5 matches DENOVO 51 21 Jan 09 11 13 has 1 matches PEAKS 1 19 Jan 09 17 22 has 1 matches PEAKS 2 19 Jan 09 17 26 has 1 matches PEAKS 3 19 Jan 09 17 29 has 1 matches DENGO 1 19 Jan 09 17 21 has 5 matches DENOWVO z 19 Jan 09 17 25 has 5 matches More information can be found about the spectra under the Info tab You will find information about the retention time where to find the spectra on the TIC graph the number of peaks and the fragmentation type f available You will also find an overview of the results that were found for that spectrum in the results files The largest panel displays the MS MS and below you will find the corresponding MS spectra under the Survey tab Information about navigating through the MS and MS MS spectra can be found above in the section describing the MS tab 4 5 Data refinement Since mass spectrometry data often contains noise and redundant data it makes sense to filter the data before analysis This will increase the quality of the results while saving time spent on database searching and de novo sequencing MS MS spectra that are mostly noise will be removed from the data When PEAKS is connected to a PEAKS Online server you will also save time by uploading smaller preprocessed data Data refinement can be done locally b
11. Rsi 1 54559 3 i689 orbSampemmML 134y Denova View SpeTwrDNLNPPEDR se oeo 1 8289 2 16397590rbSampemzML 1428 amp jHECLITECLT HGOLLECTATRO Immonium Seg Ez 1 3 04 D 14 2 so 230 08 21207 202 08 24710 N 1489 76 14717 147274 1473 75 745 39 13 3 mo 217 309 18 29914 34416 P 13 571 1357 71 1358 71 1359 66 688 37 12 4 1007 455 19 43718 427 19 47222 Q 1278 69 1260 56 1261 66 126266 639 84 11 6 11007 69330 6 528 665 29 710 32 H 1049 60 1031 09 1032 55 1033 55 52529 9 7 13608 855 36 838 25 828 96 873 99 Y 91252 89451 895 49 696 49 45676 8 8 13608 1019 43 10143 931 43 1036 46 Y 749 6 73145 73243 733 3 3 923 7 9 4405 109047 107247 1062 46 1107 49 A 586 44 568 09 S69 32 57037 29370 6 ii ao 128056 1242 56 123257 1277559 A 41629 39828 390 26 40028 20864 4 v 2 ce ca T B E m co ps pa e T gt co a a a B a T i cl ih mes ch r I umo m n m 1 lt Lr HB En ae P gt e I 2n C ea ca T e I E m a T ES T7 pq pu poc CI gt pm m m 5 m 200 400 BOO But 1000 1200 1400 1600 al 222 ox 2 9 Je Info Survey Alignment Error Map 400 600 B00 1000 1200 1400 1600 ra ce At the top of the screen you will see the peptide candidates in the Peptide Candidates F
12. The columns themselves are customizable Right click anywhere in the report and choose Toggle Column from the pop up menu The sub menu that appears shows a checkmark in each of the columns that are currently showing Click any one of them to show or hide a column These settings will apply to all your reports Confidence Scores Next to the proposed sequence candidates the auto de novo Total Local Confidence TLC and Average Local Confidence ALC confidence scores are shown The confidence scores for each amino acid that 1s confidence that the correct residue in each position has been identified are represented by color coding Red represents a very high confidence greater than 90 purple represents a high confidence 80 to 90 blue represents a medium confidence 60 to 80 and black represents a low confidence less than 60 For more detailed positional confidence place the cursor over the sequence of interest and right click Show Positional Confidence A Position Confidence Table will appear showing the confidence that each amino acid pair of amino acids are correct Sequence Tags Sequence Tag Right click on a peptide in the Peptide Candidates Frame and select Show SequenceTag QPVIDAENCHALR Ooo Sequence Tag If the score threshold is E J set at 0 0 all of the amino acids in the 550e Threshold peptide sequence will be displayed 0 0 0 1 0 2 0 3 0 4 05 0 6 0 7 0 8 0 9
13. yes O no 3 Running Protein Identification Click the PEAKS Search toolbar icon Bil or select PEAKS Search from the Tools menu The Protein Identification Parameters dialogue window will appear Enter the following parameters 117 Data Refine De nova 4 PEAKS Search SPIDER Search PTM Finder Quantification Database search ac Mass Options General Options Parent Mass Error Tolerance 0 1 Da Precursor Mass Search Type Preprocess this data on the Fly Enzyme Options Enzyme Trypsin New Enzyme Digest Rule RK Mp Delete Enzyme Find peptides that saksPythe above rule ar both ends Advance PTM Options me meroma pedere Aarete I Foti Carbamidomethylation Myristoylation fio 1984 N acyl diglyceri 788 7258 1 lt Remove Max variable PTM per peptide 3 Database Options Select Database BampleOB al species New Database Edit Database CER Datum Advanced Options PEAKS uses a hybrid search technique that requires some sequence tags to help in the search have already run de novo dont run it again Select database 7 Paste Fasta sequences j Run de novo using different parameters than the above Thermo 1 Run de novo using the same parameters as above default Validation decoy search
14. 13 2 Export Peptide Results in PepXML Format In order to export your PEAKS results file in PepXML right click on the results file that you wish to export and select Export PepXML FA Project View EAH New Project 3 E Jk Sample 1 EE OrbiSample mzxML gt FE Combine Engines Compare Results Perform Filtering Export PepaML Export Excel Export Statistics Graph Export Sequence Tag Hide Result The following window will appear A Export to PepXML Specification Select the PepxML Export Destination PepXML File Browse your computer to select the location that you would like to export the PepXML file to Then click OK 13 3 Export Results in Excel Format In order to export your PEAKS results file to Excel right click on the results file that you wish to export and select Export Excel F1 Project view di Mew Project 3 QA ITRAQ Sample yg SILAC J SILAC 1 S E SILACSample mzxML ab DEMOVO 1 19 Jan 09 14 40 Br 1 19 an 09 14 4717 v Combine Engines Compare Results Perform Filtering Export PepaML Export Excel Export Statistics Graph Hide Result 82 The following window will appear Export Excel Result Report Select the Type of Results to Export Currently Highlighted Protein and Corresponding Peptide s Complete Protein List Peptide Details Omitted C Marked Pratein s and Corresponding Peptidecs All Protein and Peptide Result s one representati
15. 1471 7454 Matched peptides shown in blue red For SPIDER MTMITDSLAV VLORRDWENP GYTOLNRLAA HPPFASWRNS EEARTDRPSQ 51 QLRSLNGEWR FAWFPAPEAY PESWLECDLP EADTVVVPSN WOMHGYDAPI 58 The Protein View collects all the peptide identifications together summarizes which proteins were present in the sample and groups homologous proteins together The same information 1s displayed in the Peptide View as in this Protein View however the results are organized to best enable us to evaluate at the protein level This view is helpful when building a summary that can be sent to a customer collaborator See chapter 13 for more details on exporting whole files or proteins of interest to an Excel file Index The top section of this view shown above behaves like an index listing each protein found in the sample Very similar proteins containing the same set or a subset of the matched peptides are clustered together To expand and collapse the full list of proteins within each cluster click the or sign respectively The table below describes the contents of the columns in the index Accession The GI accession or other unique identifier for this protein as recorded in the database that was searched Display A graphical coverage map Blue areas represent parts of the sequence that have been explained by the identified peptides Score A value from 1 to 99 representing the confidence we have in this protein iden
16. 3 4 5 had a corresponding proteins found in the database TLE ALC mjz Z Id Sequence 1 ONPQTHYYAVAVYVK 3 ANELLLNVK 1603 7993 OrbiSample mzXxML 1012 5916 Orbisample mzxML 4 RLHC 1 C 1 LNGNPEDR 1639 7307 OrbiSample mz ML 5 HSTVFDNLNPPEDR 0 69 2 1639 758 OrbiSample mzXML Now click on the Peptide View tab The following will appear in the Main processing 5 window F ID Sequence PEAKS Score 9 5 RSD Miz Z Mr Calc Delta Mass Error ppm gt o Peptides Spectrum 1 DNPOTHYYAVAVVK 99 0 0 0 802 91 2 1603 7993 0 0061 3 lt Spectrum 3 ANELLINVK 97 32 0 0 507 3 2 1012 5916 0 0062 Spectrum 3 ANEILINVK 97 32 0 0 507 3 2 1012 5916 0 0062 6 r Hit 5 HSTVFDNLPNPEDR 99 0 1639 759 Di a Eu gt _ _ _ _ _ _ _ _ _ Peptide Align Peptide Details o Immonium b b H20 a C Seq y y H20 2 1 88 04 116 03 98 02 88 04 133 06 D 5 2 87 06 230 08 212 07 202 08 247 10 N 1489 76 1471 77 E 3 70 07 Berle 309 15 299 14 344 16 P 1375 71 Id 4 101 07 455 19 437 18 427 19 472 22 Q 1278 69 1260 56 5 74 06 556 24 538 23 528 24 573 26 T 1150 64 1132 62 6 110 07 693 30 675 28 665 29 710 32 H 1049 60 1031 49 7 136 08 856 36 838 35 828 36 873 39 Y 912 52 894 51 8 136 08 1019 43 1001 43 991 43 1036 45 Y 749 46 731 45 9 44 05 10
17. don t run it again gt Run de novo using different parameters than the above orbisample MI Run de nowo using the same parameters as above default validation decoy search hd 112 Click OK This will launch PEAKS Protein ID and when completed the results will appear as below ID Sequence PEAKS Score RSD Miz 4 Peptides Spectrum 1 Peptide Align Peptide Details Immonium b b H20 188 15 215 15 198 14 hart view Protein view Peptide view Denovo View 601 388 A 1 PLDMDIGVSEATR 99 0 0 0 388 oo Spectrum 3 1 M NWOQHK 2Z IGGL oo Spectrum 4 TLLLFISR amp 543 QD 44758 2 oo Spectrum 5 D 1 WENPGVTQLNR Spectrum M 1 SGIFR 4887 QD 427727 2 o gt Spectrum 7 M i SGLFR 488 og 427727 a Spectrum 8 V T IDEDQPFPAVPK 2 Seg Pri alc 160081 76 2042 00 073 5299 1571 7815 ama 4602 Ba3 4602 1628 8652 0 0198 12 1407 5ample iTRAQ mzXML Delta Mass Error pprm File RT 0 056 35 0785 Sample iTRAQ mzxML D 0864 42 3222 5ample iTRAQ mzxML o OU 0325 36 91985ample iTRAQ mzxXML 0 02 13 97975ample iTRAQ mzxML U 0208 24 3872 5ample iTRAQ mzxML Es Y af D a 0 0208 24 3872 5ample iTRAQ mzXML D 386 68 047 48 719 38 1 102 06 1255 63 1237 62 1227 63 1272 66 476 25 458 24 459 22 460 22 238 62 d m 200 dh 1 2X 2v Info S
18. found under the spectrum view on the right hand side 56 To zoom either on the X or Y axes select the Zoom X or Zoom Y buttons respectively and then use the wheel on your mouse to move around the graph Select the Slide X button and then use the wheel on your mouse to move around the graph You must ensure that you are sufficiently zoomed in on the X axis to use the Slide X button Selecting the 1 1 button will bring you back to the original image where you can see the entire spectrum on the screen You can use the profile atl and peak ull buttons to switch the spectrum view from profile mode to peak mode and vice versa The scrollbar on the left acts to increase and decrease the intensity of the peaks where the scrollbar on the right acts to zoom in to display the monoisotopic peaks Finally at the bottom of the screen is the Spectrum Alignment Frame which is used as a tool to navigate the Spectrum View frame A blue bar along the horizontal axis of the alignment view indicates the range of the spectrum view in the Spectrum View Frame This frame will show you how the proposed ions align with the spectrum By default the Spectrum Alignment Frame displays b ion and y ion The b ions are shown right to left in blue while the y ions are shown left to right in red Peptide Details Click on the Peptide Details tab The following window will appear Peptide Align Peptide Details S
19. 0 0 507 3 2 1012 5916 0 0062 6 0879 0rbiSample m 0 365 0 781 97 37 zx HSTVFDNLPMP a Spectrum 4 96 69 0 57 547 59 3 0 0 0 0107 6 551 1 Orbisample m T 1 347 0 777 96 69 i Spectrum 5 99 0 0 14 820 89 2 0 0 0 0065 3 9455 Orbisample m 1 428 0 787 99 0 gt Oa 5 Peptide Align Immonium b b H20 a C Seq y y H20 z zi y 2 E 1 110 07 138 07 120 08 110 07 155 09 H 14 2 60 04 225 10 207 09 197 10 242 13 5 1503 71 1485 70 1486 68 1487 68 752 35 13 5 3 74 06 326 15 308 14 298 15 343 17 T 1416 68 1398 66 1399 65 1400 65 709 32 12 4 72 08 425 22 407 21 397 22 442 24 Y 1315 63 1297 62 1298 60 1299 60 558 31 11 5 120 08 572 28 554 27 544 29 589 31 F 1216 56 1198 55 1199 53 1200 53 508 78 10 6 88 04 687 32 669 30 659 32 704 34 D 1069 49 1051 48 1052 46 1053 46 535 25 9 7 87 06 801 37 783 34 773 36 818 38 N 954 46 936 45 937 44 938 44 477 73 8 8 865 10 914 44 896 41 886 44 931 46 L 840 42 822 41 823 39 824 39 420 71 7 9 70 07 1011 49 993 48 983 49 1028 52 P 727 33 709 33 710 31 711 31 364 17 6 E 10 87 06 1125 53 1107 52 1097 54 1142 56 N 630 28 512 27 513 26 514 26 315 64 5 11 70 07 1222 59 1204 58 1194 53 1239 61 P 516 25 498 23 499 20 500 21 258 62 4 v T I T e E Y 5 T o o z e a F F a zt amp x 212 g Sl 7 n a 1 ef ts gt 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 aur 1d 2a zw D 100 200 300 400 500 600 700 800 900 1000 1100 1200 13
20. 1 0 d amp Sequence Tag Increasing the Score Threshold oo SS wil display a mass in square ate La brackets if the amino acids do not satisfy the score threshold Modifications Consider the following sequence P L1 C 1 5FTDAENVQALAR The number in square brackets refers to where a modification may occur If you forget what modifications you selected before running de novo click to the Properties tab Denovo details Test 6 The fixed modification 1S set to 1 58 005478 CKW In the sequence above the Parant Mass Error Tolerance 0 1 da modification has been made to the W as well as the C Fragment Mass Error he m Enzyme Trypsin The colors assigned to the 1 follow the same i ia fid h l ds th Semi enzyme False confidence scores as the amino acids themselves Fixed Modification 1158 005478G CkW Refer to the above section on Confidence Scores for Max variable PTM per peptide 2 more information on color coding Report peptides Prepracess data False 48 Ion Table Frame Immonium b b HzO a C Seq y y H2O z z viz E 1 33 04 115 03 93 02 33 04 133 06 D 14 2 8 0 23 0 azor 2208 2710 N 14976 i737 i4z74 14875 74939 13 3 7o sz 39is 29914 3416 197i 1597571 13871 13966 68937 12 4 100 4519 43ris 49 4222 Q izes 126056 126166 126266 63994 11 74 06 6 amoo 69530 67528 6529 7032 H 104960 105149 10255
21. 103355 59229 9 7 13608 8535 85935 8836 873 59 Y Sins esasi 89549 59549 45576 8 8 19508 391 43 9 ao ior 107247 10246 110749 A SS amp 4 see2s seo3z smoar 2397 e i 44 12056 12256 123257 127759 A 46 9 39828 39926 40026 20964 The Ion Table shows the proposed 1ons with their corresponding masses To add additional ions to the 1on table see the instructions on page 95 If an 1on is found in the corresponding spectrum it must first pass two criteria before being displayed in a specific color blue for N terminal ions and red for C terminal ions It must be found within the mass error tolerance chosen by the user and must have an intensity of greater than 2 of the ion with the greatest intensity Spectrum View Frame y10 y11 H20 b11 y8 28 y9 H20 b9 b10 b11 H20 2 2 y3 H20 b3 y y13 b2 H20 y3 b5 NH3 POTHY Y Av NH3 y4 y5 y8 a0 H20 b6 H20 POTHY Y b7 POTHY YA b8 b9 NH3 all NH3 b13 H20 gt yl a 100 200 300 400 500 600 700 EF MEDIE 2 co e e 900 1000 1100 1200 1300 1400 1500 1500 1700 The Spectrum View Frame is found below the Ion Table and displays a graphical representation of the spectrum The peptide that corresponds to the spectrum in the Spectrum View Frame is displayed in the Input Sequence box Use the drop down to select other peptides that have the same ID Scroll
22. 2 Peptide Index Search Engines PEAKS omssa only 0 PEAKS only 71 Protein Score Distribution Chart Peptide Score Distribution Chart Apply InChorus Protein Number Venn Diagram XTANDEM The top two charts the Protein Score Distribution Chart and the Peptide Score Distribution Chart are in the same format to those that are seen in the Chart View of a PEAKS Protein ID search page 61 More information about the inChorus Protein Number Pie Chart and the inChorus Protein Number Venn Diagram can be found on page 72 3 8 Perform a SPIDER Search In this example spectrum 6 has not been identified with database searching tools In order to gain more information from our data we will run a SPIDER search next For more information about the SPIDER search refer to page 63 1 Click on the OrbiSample mzxml file 2 Next click the SPIDER Search toolbar icon 1 Or Select SPIDER Search from the Tools menu 3 Enter the settings as shown 29 FN SPIDER Search t Data Refine De nava PEAKS Search SPIDER Search PTM Finder Quantification a pi SPIDER Search C serana j Segment Match Non gapped Homology Match Homology Match Mass TE Da 0 1 Leucine equals Isoleucine Lysine equals Glutamine PTM Options Mame Mona mass Fixed Modification LICFUIIMACcION IH
23. AP FOV CC EE IT 12 3 3 Perform Data Refinement 0e00000000seossooseosessesosossessesssossessesosossessessssssossessssssosses 16 5 4 R n De novo Sequencllg ssosecssacssecisesvensusadehcucstsenedueenuteniansvendesalihaveceeensddansecwddangvesess 17 3 5 Run Protein Identification 00000000e00sooseosesssoosossessesosossessesssossessesssoosessessssssesseo 19 30 Run PIM EWImdeE 2 oreet usie aaa ie coo Coo Pes Doe Dee o eco a es Pe oue E Oeo Pei e ido 22 3 7 Run ammchorus SedEelIrzau ise ceecktu dace tuere do as ca eoo a Roi Nea Oe Focus peas Coca CELL e OPER Eo EOM ER DEDE 24 3 5 Perform a SPIDER Search iie eren rare eo ev esr P Eso ge E NER R Ce Dn Oen e EBe e UR Na 29 t NGO AG GALA rea STI et 32 LL Data QNIN TT DU 32 42 Data BEL RES SLUT P 32 PORNO Nbre 33 Aalen DAA oeiras e 33 Br k r Dalier i a Aa 33 Shimadz0 Bc 33 Applied Biosystems DACA sssscscescisecacsteassvesersececkeenssreckesscecateasavevevvececneensseseteaisecaaes 33 varian PACA wos OUI T Mt 33 Waters Micromass MassLynx Data ssccccccsssssssccccssssssccccccsssssccccccsssscccoes 33 ABI 4700 or bic ERE 34 4 5 Load a New Ele iie ihe seed eec iive eene Era Qoa been e sauce covebeletecsecsecctsvelevacuescecacesedcceseesks 35 44 Create a NOW PrOJect ia uet teen tens ra adonca NT ataoa PIERII DH EM ERES 36 T 5 ODnDemL E PONCE E ee ee su v ee EeePC Code E CE AE 38 4 6 Changing the
24. Applied BiosyskEe 442 225 C ig anywhere MEM Applied Biosyste 450 2752 C gp nywhere Applied Biosyste 227 12698 C Anywhere unimad Delete PTM New PTM Edit PTM You will now be able to add various modifications to your amino acid sequence For example enter A M then select Oxidation on M from the PTM list and click the PTM button Then click the Calculate button You should see the following Input Sequence or Mass Daj 9 b ion 2 y ion non specific AP o M gt Peptide Mass Charge Me Mass To view additional PTMs from the unimod list select the unimod box You can delete a PIM using the Delete PTM button You can also edit or create a new PTM using the Edit PTM or New PTM buttons respectively You can also use the advanced options menu to add a water or proton to your sequence The Back button will remove the last amino acid or modification that you have added to your sequence or click on the Clear button to start again 127 19 About Bioinformatics Solutions Inc BSI provides advanced software tools for analysis of biological data Bioinformatics Solutions Inc develops advanced algorithms based on innovative ideas and research providing solutions to fundamental bioinformatics problems This small adaptable group is committed to serving the needs of pharmaceutical biotechnological and academic scientists and to the progression
25. G CID n2 RAW 9 CID o3 RAW Jl CID ETD DENOVO 151 19 Jan 09 19 47 tp PEAKS 31 19 Jan 09 21 21 0 ESTEEEIERBEIMEDEN 9 CID ETD 01 RAW sedis s After selecting the Compare Results off CID_ETD_O RAW Compare Results 9 CID ETE DS RAW Perform Filtering function for PEAKS 11 PEAKS 22 and Export PepXML PEAKS 32 a new entry will appear in the Export Excel Project View Frame called LE Compare Export Statistics Graph PEAKS 11 22 32 Hide Result Note that the results window that appears will contain De novo View Protein View Compare View and Compare Chart tabs In the Protein View all proteins found by each of the in the result of PEAKS 11 PEAKS 22 or PEAKS 32 are listed in the first column For each protein the scores in different database search results are displayed 79 PEAKS 32 20 Jan 09 09 21 x Compare 11 22 32 x Se Accession PEAKS 11 PEAKS 22 PEAKS 32 Mass Description DB Search i Q93615 ETFA_CAEEL 99 14 34454 562 Probable electron transfer flavoprotein subunit alpha mitochondrial OS Caenorhabditis elegans GN F27D4 1 PE 2 SV Te Q91717 CO2A1 XENLA 88 25 142263 17 Collagen alpha 1 II chain OS Xenopus laevis GN col2a1 PE 2 SV 2 ie Q17967 PDI1_CAEEL 99 15 53436 16 Protein disulfide isomerase 1 OS Caenorhabditis elegans GN pdi 1 PE 2 SV 1 O02056 RL4 CAEEL 99 14 38659 105 605 ribosomal protein L4 OS Caenorhabditis elegans GN rpl 4 PE 1 SV 3 0 Q6P4Z
26. NO to copy this URL to system clipboard You can start your FTP client software manually After download the database you don t need to do decompression Put the Files in the directory that PEAKS can access 101 If you would like to invoke your default FTP client 1 The URL Fo download the database has been copied to system clipboard software and download LD Please start your FTP client software to download the database automatically click Yes If you select No the following window will appear telling you that the URL will be copied to your system clipboard Click OK Open your Internet Explorer and paste the URL into the address bar A file download window will open Click Save Repeat Step 11 taxdmp file Please not that you do not have to decompress the taxonomy files 12 Now that you have downloaded the taxonomy files you must tell PEAKS where to access them by clicking the Browse button and selecting the file 13 To save the database to your Database List you must click the Add Update button before clicking the Ok button Delete a previously saved database If you would like to delete a database file select the database that you wish to delete and click on the Delete Database button Set a database as default Select the file and click the set as default button which is located to the right of the Database List This database will now be used by PEAKS when you run PEAKS Pro
27. NOVO Sequeneome ReSUlfs iioi tevdeeu epa seat ea aque EX EER EE Ue voveo a sur Ebo e Va uu tasane renia 46 Peptide Candidates Frannie oer n erre a ebo eee o ee aeo E e Nueva Nue ea e vega i 47 lop Table dli S 49 Spectrunr View EEAII 65 io enr He ERE EEUU a aa Ea Ea eaaa 49 Spectrum Alignment Frame cccccccssssssesessssscvssscccccvossovssssesssseessssssoovesecscoosses 50 SOULE V CY SCIll crate nr Dania Weit Win Hn Id ISP 50 Error VEAP sate cccececss scatesdeeceuacec sackets iewetectetscueedsexeuccnsssceuusvexesastesueuseusexeuacacesenaueeveaes 50 To Database Scare ascen EAM M 51 7 1 Setting up Protein Identification Parameters ecce eee eee eee e eere ne 51 Mass IUUD T I LO ceca cdeteaenvoseess 52 BETZ y ICO PU OUNS ameinasa M 52 General Bri e T 53 PIM SIDE HP 53 Database O nit 54 Advanced POMS e riier teteet ioa e de iE eae eu EE EEE aE EVE DEI I ESTE iaa 54 72 Protein Identification Results erede eser ove ve eue pE Ep aee TN eo Y ve eee e a Eoo eee EY eseese ei 55 Peptide V TOW 4n en eetete iere v Feet p ive oves ees uae e N ved ane RE VEU veio EE 55 Iura ET UU Tm 58 AEE VC Mee sec Ea aA E Er Ri 61 o SPIDER SedECIE tire EE HE e E E 63 8 1 Setting up SPIDER Paramaeeters ce eee ee e eee eee eese e eee es eee eese eese soos eee eese 63 Query iL rc 64 GemCr all POMS eM 65 PTM ODUOHS sani
28. Protein Filters 74 Filter Options The filters cascade through each view in the multi part report For instance removing a peptide from the database search results the Peptide View list will cause the de novo sequence for that peptide to be removed from the De novo View tab but will not affect the Protein View list Filtering out a protein by mass for example will remove it from the Protein View list and will remove all peptides associated with that protein from the Peptide View list as well as from the De novo View list The manner in which the De novo View is linked can be specified by the user using the options in the Filter Options frame See the figure below for more information Option Selected What De Nove View Displays How Filters are linked Options De nove view shows peptides that could notbe explained by peptides from the Peptide View Spectra Y enplaimead tuy database Filtering out removing a de novo result does not affect what is displayed in the Prote n View tab or Peptide Yiew tab 2 De novo view shows all peptides that are not filtered Remove ce novo peptides with na database hits Spectre expinined hy Ge npo Uptlorns De novo view shows peptides that could not be explained by peptides from the Peotice View Filtering out removing a de novo result also 8 Do novo view shows all S xe removes that spectrum peptides that are
29. The panel along the bottom allows you to narrow in on the peptides that you would like to examine You can specify a particular scan number range m z range or intensity range Click the Apply button to change the 3D view to your specified values Check the Auto button and click Apply to the default numbers Browse D AderbyServerlserverDBlTRAQ Sample Notes Description Type and organization of project Basic Project Give your project a name such as 1TRAQ Sample Then click Next The following window will appear Give your sample a name such as 1TRAQ 1 A New Project Steps Sample Properties Cli ck the ec A dd a file for 1 Project Properties Give this sample a name iTRAQ 1 2 Sample Properties Select Files this sample button and select the file C PEAKS 5 Data iTRAQSample mzx ml Click OK to add this to the list of selected files J a Name Date Modified Add a file For this sample Remove file From list Clear list Sample Notes Description For cases where you want to add another sample to the project select Add 99 E OET P and 1 pdt these last two steps In our case we are not going to add any more samples so we can just click Next amp New Project Steps Sample Properties 1 Project Properties Give this sample a name iTRAGQ 1 bul 2 Sample Properties Select Files cu Marne Size Date Modif
30. are designed to work in the same way as options screens for the original programs For help in setting search parameters for each program please refer to that program s user manual For help with PEAKS Protein ID please refer page 51 Importing Existing Results PEAKS inChorus reads X Tandem xml files OMSSA omx files Mascot dat files and Sequest srf files When importing third party results files please make sure that the scan number model in the results file is consistent with the one in PEAKS PEAKS uses original data information to compute the inChorus score When you run X Tandem search with command line you need to turn on the option of w in order to export data information into X Tandem xml files To import existing results check the Import Result checkbox and select Import Result The following window will open InChorus Search Import Result Database PEAKS Protein ID Define a common database For importing search result Database to search swiss Tandem Taxonomy selections lall species omssa Sequest Import Search Result v import Result NDS i Import Tandem xml File Import Omssa omx File Import Mascot dat File Import Sequest srf File i Although it is not necessary for the various search engines to use the same database in an inChorus search it is necessary to have a unified database for an inChorus search that includes imported results Select the database that
31. can select De novo PEAKS Parameters or SPIDER Parameters The parameters that you have saved within these categories will be displayed below in the list Select the parameter file that you would like to view 104 Creating a new parameter file If you would like to create and save new parameters you can do this when before you set up auto de novo sequencing see page 45 PEAKS protein ID see page 54 or SPIDER see page 66 These references will provide you with an explanation of all of the parameters Deleting a previously saved parameter file If you would like to delete a parameter file select the file that you wish to delete and click on the Delete button Viewing a previously saved parameter file Selecting a file will display the details of that file below For an explanation of the parameters please see the pages listed in the Creating a new parameter file section above 105 15 PEAKS Quantification Many approaches to protein quantification using mass spectrometry data have been described in the literature In terms of their implementation most of them can be classified into three protocols e MS Quantification based on the relative intensities of extracted ion chromatograms XICs for precursors within a single data set This is the most widely used approach which can be used with any chemistry that creates a precursor mass shift For example ICAT and SILAC e MS MS Quantification based on the relative int
32. different database search results are displayed PEAKS 32 20 Jan 09 09 21 x Compare 11 22 32 x Sequence PEAKS 11 SpectrumId PEAKS 11 MZ PEAKS 11 Score PEAKS 22 Spectrum Id PEAKS 22 MZ PEAKS 22 Score PEAKS 32 Spectrum Id PEAKS 32 MZ PEAKS 32 Score 9 LDATVHGEVSSK Spectrum 6875 621 82 16 68 Spectrum 38495 621 82 37 5 A 5 ATGVLYDYVNK Spectrum 6875 621 82 4 63 ca B NwXXNZMZK Spectrum 8m73 bea E yy 5 EwNZNZEN Spedrum2 488 73 Ra LLLI 5 D 3890 513 31 42 88 Spectrum 10175 13 64 Spectrum 29859 513 31 98 9 8 APPAKVSK Spectrum 6964 513 31 4 08 Spectrum 10175 8 49 z SPQ 2 SGTN 2 KK Spectrum 4 474 74 5 56 ExsNZMZNK wedums haa 28 QEYDESGPSIVHR Spedrums o2 wea O OOOO Beedrm2962 0624 6794 B TKENZLGRSAMSNR Spedum6 paa pz T LL OG __ DFN 2 VEYIQRGGLR Spectrum 5282 784 39 121 The Compare Chart provides Venn diagrams and pie charts for proteins and peptides to illustrate the comparison of results that were generated by each protein ID search For more information on the Venn diagrams and pie charts in PEAKS s Chart View refer to page 61 i Protein Number Pie Chart m gt a Protein Number Venn Diagram PEAKS 11 PEAKS 22 PEAKS 32 only 23 7 PEAKS 11 only 25 1 PEAKS 22 only 34 7 PEAKS 32 Peptide Number Pie Chart PE
33. error on the fragment daughter ion PEAKS will allow for in its analysis As an instrument has previously been selected PEAKS will provide suggested error tolerances Type a tolerance in the textbox Again new PEAKS users should try setting this a little higher than past experience may suggest Enzyme Options Enzyme Tell PEAKS what type of enzyme was used to digest the sample Choose from a dropdown list of enzymes or if your enzyme is not in the list click the New Enzyme button You can then input the name of the new enzyme Digest Rules Enter the amino acid that is found at the end of the peptide Put set brackets j around a residue to denote any amino acids except for those that are within the brackets Select the Advanced button if your digest rules are more complicated Select the radio box Select peptides that satisfy at both ends if you require that your peptide was cut by the enzyme you chose at both ends PTM Options Selecting fixed and variable PTMs The PTM Options list tells PEAKS what types of post translational modifications to include in its analysis To view additional modifications select the Show unimod box If a desired PTM does not appear on the list or is different than what is listed select the New PTM button and the PTM Editing window will open Fill in the information pertaining to your PTM To select a PTM as Fixed or Variable drag the PTM into the Fixed Modification or Variable M
34. from the drop down menu If you selected the General setting in the option above the instrument names will also be general however if you selected a particular vendor the vendor specific instrument names will be displayed If you do not see the instrument that you used you click on the Add Instrument button to create a new instrument 3 Finally browse your computer to locate the file to be processed and click open The file will now begin loading F3 New File X Open Files 1 Select Manufacturer Project name New Project 1 2 Select Instrument Select vendor Thermo Scientific d Seteck pels Select instrument LTQ FT Ultra Hybrid FT Trap Add Instrument Lookin sampleData m ESSE t cie Es zia Recent Items Desktop Documents E i j ear m Computer x Network File name Open Files of type raw Thermo Xcalibur raw File Cancel 35 4 4 Create a New Project 1 To create a new project select New Project from the file menu or using the New project 2 Create a name fem vem 3 icon le on the toolbar The Project Properties window will open for your project and click Steps Project Properties browse to 1 Project Properties Project Name Project 1 select the 7 location of the data for that project You can use the notes and description box to remind yourself of information specific to the project The Sample P
35. if the setting 1s too high you can determine the highest optimal value for your own computer 23 Installation for Windows Users Note If you already have an older version of PEAKS installed on your system please uninstall it before proceeding 1 Close all programs that are currently running 2 Insert the PEAKS 5 disc into the CD ROM drive If loading PEAKS via the download link skip to step 4 after downloading and running the file 3 Auto run should automatically load the installation software If it does not find the CD ROM drive and open it to access the disc Click on the PEAKS Studio Installation exe 4 A menu screen will appear Select the top item PEAKS Installer The installation utility will begin the install Wait while it does so When the PEAKS 5 installation dialogue appears click the Next button YE Peaks Studio 5 0 En Introduction a introduction InstallAnywhere will quide you through the installation of Peaks Studio 5 0 tis strongly recommended that you quit all programs before continuing with this installation Click the Mext button to proceed ta the next screen If vau want to change something on a previous screen click the Previous button You may cancel this installation at any time by clicking the Cancel button InstalAnmwhere by Macrovision Cancel Previous l 5 Basic system requirements will be presented Please note that while the minimum requirement is 1 G of RAM
36. locations Default Output Directory Output Directory Browse C derbyServer Project Folder Browse C derbyServer serverDB You can also change the location of you projects on a project by project basis by selecting a new Project Location when setting up a database as seen below amp New Project Steps Project Properties 1 Project Properties Project Mame Project 1 2 o Project Location CiPeaks Studia 5 0 derbyServerlserverDB Browse 4 7 Orienting Yourself Project View Panel y Project view This frame appears in the upper left hand Sip CuPeaks Studio 5 0 derbyServer serverDB New Project 1 corner displays the organization of a l Sample 1 particular project if applicable or simply of a eee Jor biSample mzsML data file Use the and boxes to expand and collapse the project in order to access the data file that you want to analyze Make sure the data file to be Tasks Properties analyzed 1s selected Selection Details 9ProteinMix FT RAW Properties Panel Total MSI Spectra 1283 PEAKS reads and tracks information about the Total MS MS Spectra 3712 experiment for use in the analysis and for future iE ENS Fragmentation Mode CID CAD IRMFPD v reference Once the data file has loaded click on the MS Scan Made FT ICR Orbitrap properties tab in the bottom left hand corner If any MS MS Scan Mode Linear Ion Trap information cannot be found in the file PEAKS will MS Scan C
37. mzXML gy DENOVO 2 12 Jan 09 14 24 i DENOVO 1 12 Jan 09 14 19 W PEAKS 1 12 Jan 09 14 20 OW Combine Engines oo MI XTANDEM 4 1 C Result LM OMSSA 5 12 ee Perform Filtering Export Pepe Export Excel Export Statistics Graph Hide Result Possible Filters Selected Filters Edit Filter The filters are grouped into three basic types to reflect what they act on De novo filters act to remove proposed de novo sequences Peptide Filters act to remove peptides found in the database from the report and Protein filters act to remove proteins from the report To see the available filters for each level of filtering double click on the appropriate folder in the Possible Filters frame See the options in each folder below DO Be Move Filters fer Peptide Filters fae Protein Filters d Td Filter e ID filter Acc filter Sequence filter a Sequence Filter eM ID filter n Score TLC filter A Score Filter eM Mass Filter 4 Score ALC filter e miz Filter e Score filter 4 Rank filter 4 Charge filter 4 Coverage filter 4 miz Filter Mr calc Filter 4 Query filer ie Charge filter e Delta Mass filter see 4 Selected Filter Mass filer Errorippmy Filter woe Desc Filter se File filter e File Filter so Length filter 4 Length filter RSD filer 4 Engine score filter Choose a filter from the Possible Filt
38. not filtered from consideration in Remove de novo peptides the Peptide hei tab and with no database nits Der Protein View tab explained by de mira Options De nove view shows peptides thal could not be explained by peptides from the Peptide View Again filtering out removing a de novo result also removes that spectrum from consider ation in the Peptide View tab and Protein View tab erated by e De novo view shows all P peptides that are not filtered v Remove ce novo peptides with no database hits Spectra explained by de novo 75 Parameter Options Filter sets can be saved and re used between sessions by clicking the Save Parameter button that is found at the top right hand corner of the Filter Parameters window You may prepare your results the same way each time in which case it makes sense to set up a filter that will be automatically applied each time we load a report Select a filter from the list of saved filters from the dropdown menu found at the top right hand corner of the Filter Parameters window Click on the Set saved as default button This filter will be displayed in the Parameter Options frame as seen below and will be applied automatically just after a report is loaded Be careful if your default filter is very stringent it can sometimes parameter options remove everything To remove a default filter press the Clear esses Default Button at the bottom of t
39. of drug discovery research The company founded in 2000 in Waterloo Canada comprises a select group of talented award winning developers scientists and sales people At BSI groundbreaking research and customer focus go hand in hand on our journey towards excellent software solutions We value an intellectual space that fosters learning and an understanding of current scientific knowledge With an understanding of theory we can focus our talents on providing solutions to difficult otherwise unsolved problems that have resulted in research bottlenecks At BSI we are not satisfied with a solution that goes only partway to solving these problems our solutions must offer something more than existing software The BSI team recognizes that real people will use our software tools As such we hold in principle that it is not enough to develop solely on theory we must develop with customer needs in mind We believe the only solution is one that incorporates quality and timely results a satisfying product experience customer support and two way communication So then we value market research development flexibility and company wide collaboration evolving our offerings to match the market user s needs Efficient and concentrated research development customer focus and market analysis have produced PEAKS software for protein and peptide identification from tandem mass spectrometry data RAPTOR and PROSPECT Pro software for threading based 3D p
40. of the particular peak under the spectrum view on the right hand side see the box highlighted in red below You can use the profile ill and peak An buttons to switch the spectrum view from profile mode to peak mode and vice versa 570 580 590 600 610 620 630 640 650 660 lt 4 li 1 1 2 2 OrbiSample mzXML ms 1 RT 1 5 scan 9 TIC 5 23E7 565 5469 0 05 40 The MS MS tab gives t OrbiSample mzXML we detailed information gt 695 842 507 32 about each tandem p gt e793 spectrum B gt 706 623 802 91 2 id 1 N 2 NPQTHYYAVAVVK 0 6 C DNPKTHYYAVAVVK 0 6 DNPAGTHYYAVAVVK 0 6 DNPQTHYYAVAVVK 0 6 L DNPGATHYYAVAVVK 0 6 gg DENOYO 51 21 Jan 09 11 13 200 400 600 800 1000 1200 1400 1500 TN 1 1 2 2 OrbiSample mzXML ms 22 mz 802 91 2 2 RT 0 035 TIC 1 79E7 Info Survey 200 400 600 800 1000 1200 1400 1600 RUN i22 OrbiSample mzxML ms 1 RT 0 01 scan 1 TIC 1 27E8 0 0 Each of the spectra in the data file will be listed in the left most panel under the name of the data file Clicking on one of the spectra will display the results that have been generated for that spectrum in the top right hand panel as seen in the example above Before any results files have been generated the top panel will look like this Ea Li a no result mo result aana IR FS B d 547 50 Info Survey Selected MS MS OrbiSample mzxML 802 91 2
41. of these peptides are very close and that they have similar retention times in the LC column The units here are m z values in Daltons For retention time we use whatever units are recorded in the data file usually minutes or seconds Precursor Charge Correction Since a mass spectrometer measures mass to charge ratios we must know the charge on a peptide before we can determine its mass The standard method of finding the charge is to look at the spacing of the isotope ladder in the survey scan However many Ion Trap instruments do not have enough resolution for this So PEAKS will look at the MS MS data to determine if it s charge 1 2 or 3 For data where the survey scan is available PEAKS will examine the precursor ion s isotope distribution to confirm or correct the charge assignment Type in the boxes to set a range of charges Only spectra that fit in this range will be considered for analysis Filtering MS MS Scans Scans of contaminants and electrical noise should not be included in analysis Removing them from the data set will save time and reduce the risk of random matches to the database PEAKS offers an effective tool for removing these low quality MS MS scans Type in the boxes to set ranges of retention time and m z ratio Only peaks between these values will be considered for analysis Additionally PEAKS examines the MS MS spectrum to determine its quality The quality filter is based on four characteristics signal to noise
42. peptide Find peptides that satisfy the above rules at both ends Add Update 97 You must click the Add Update button for the changes to be saved Your new enzyme will now appear in the Enzyme List where you can access it later If you wish to delete an enzyme that you created select the appropriate enzyme and click the Delete Enzyme button Note For information on defining new enzymes on the fly for PEAKS de novo or PEAKS Protein ID see pg 45 or pg 53 respectively PTM Configuration From the Configuration window select PTM from the left hand to change your PTM configuration Built in PTMs The built in PTMs within PEAKS are listed in the PTM List To see additional built in PTMs from the Unimod library click the Show unimod box Clicking on one of these built in PTMs will display the information listed about that PTMs in the PTM Details panel Note that you cannot delete or change the details of a built in PTM and therefore the Delete PTM button and the PTM Rules panel will be grayed out Create a new PTM Click on the New PTM button Now simply enter the information about your PTM in the PTM Details panel Name this name will appear in the PTM list for future use after it 1s saved Monoisotopic mass the mass that the residue gains or loses as a result of the PTM Neutral loss mass the mass that the modified residue loses as a result of fragmentation Ex 28
43. put all our databases in a folder c peaksdatabases The folder c my documents databases wouldn t work because it contains a space between my and documents Using spaces in the database file name causes the same problem So after you download and extract our database you should call the database file ncbinr fas or ncbi nr fas rather than ncbi nr fas Instrument Configuration From the Configuration window select Instrument from the left hand to change your instrument configuration Built in Instruments Select the manufacturer of your instrument from the drop down list The names of the instruments will then appear in their vendor specific formats Select your instrument and you will be able to view the information on your instrument in the Instrument details panel below You can also select General in the manufacturer list and the instruments will be listed in a general format Note that you cannot delete or change the details of a built in instrument and therefore the Delete PTM button and the Instrument Details panel will be grayed out Create a new instrument Click on the New Instrument button and the following window will appear Configuration In the Instrument Details dcs panel create a name for Manufacturer General v 1 e your instrument e Parameters FT trap etd FT trap pqd FTMS FTMS ecd FTMS ecd cid Next f
44. right to left in blue while the y ions are shown left to right in red Survey Scan Info Survey Alignment Error Map Ld 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 TN IDE A OrbiSample mzxML ms 1 RT 0 01 scan 1 TIC 1 27E8 Clicking on the Survey tab will display the corresponding precursor ion spectrum The buttons that appear in this section are the same as those that are explained above in the Spectrum View Frame section Error Map Info Survey Alignment Error Map 0 100 200 300 400 500 600 700 800 200 1000 1100 1200 1300 1400 1500 1600 1700 Click on the Error Map tab The m z ratio 1s displayed on the y axis and the error 1s listed on the x axis 1n Daltons The Error Plot displays the confidence that 1s assigned to each 10n The most confident results lie on the centerline Clicking a cell or column in the Ion Table highlights the corresponding points on the error plot and corresponding peaks on the spectrum 50 7 Database Search 7 1 Setting up Protein Identification Parameters 1 In the Project View Frame select the data file s or project containing the spectra that you wish to identify using database search 2 Click the Protein Identification toolbar icon Eil Or Select PEAKS Protein ID from the Tools menu Database Search Save Parameter The Protein Identification Mass Options General Options Parameters Parent Mass Error To
45. search engines is useful both for finding new proteins and confirming others You can perform and inChorus search using PEAKS Protein ID X Tandem OMSSA Mascot and Sequest For this example we will be performing a local search using the X Tandem and OMSSA search engines If you have not already set up your search engine preferences see page 93 for more instructions 1 Click on the orbisample mzxml file 2 Then click the inChorus Search toolbar icon D Or Select inChorus Search from the Tools menu 24 The inChorus search window will open Check the PEAKS Protein ID box and select the name Enter the following settings InChorus Search Tools PEAKS Protein ID 2 Tandem OMSSA F Mascot F Sequest Import Result EJ FEAKS Protein ID Database Search Mass Options General Options Parent Mass Error Tolerance Precursor Mass Search Type Preprocess this data on the Fly Fragment Mass Error Tolerance Da Monoisotopic 7 Average Max Missed Cleavages if Enzyme Options PTM Options Mano mass Residue site Add Fixed gt ci Fixed Modification m Iodoacetic acid derivative re E Mannosylation 162 0528 Dearmidation 0 984 No ehydration 18 0106 T5 HG Add variable gt Variable Modification imethylation 28 0313 CKRHDENQ Add Variable gt C P TEE dera lt Remove Deamidation Flavin adenine 783 1415 CHY Farnesylation 204 187
46. search protein identification Please note that version upgrades of PEAKS as well as upgrades to the databases may result in small changes to the results screenshots in this chapter 3 1 Create a Sample Database Before running the walkthrough data you need to set up a database So that this can be a quick process we have provided you with a sample fasta database called SampleDB fasta in your PEAKS program folder C PEAKS Studio 5 0 Data Click on the configuration toolbar icon or select Configuration from the Tools menu Select Database from the left hand side of the window Under Database Details enter the following information Database Details FASTA Format database LIniPratKB Swiss Prak hal Basic Options Database name Sample DE Download Database Path CiiPeaks Studio 5 0 DatalSampleDB Fasta EST database You do not need to change any of the other information listed Click the Add Update button and then click OK 3 2 Create a Project This will be a rather simple project as it will only contain one sample however the same process will be used for projects with multiple samples and files Click on the Create new project button amp 3 or select New Project from the File menu The following window will appear amp New Project Steps 1 Project Properties ET amp New Project Steps 1 Project Properties 2 Sample Properties no Project
47. subscribers of BSI s Maintenance plan at BSI s then current rates Technical support is available by phone fax and email between the hours of 9 am and 5 pm Eastern Time excluding statutory holidays 10 Governing Law This Agreement shall be governed by and construed in accordance with the laws in force in the Province of Ontario and the laws of Canada applicable therein without giving effect to conflict of law provisions and without giving effect to United Nations Convention on contracts for the International Sale of Goods 130 For technical support issues not found in this manual please contact either your Sales Representative or any of our support service resources email supporta amp bioinfor com tel 519 885 8288 fax 519 885 9075 online www bioinfor com peaks E Lb d di
48. the preferred configuration is 1 5 G of RAM Click Next 6 Read the license agreement If you agree with it change the radio button at the bottom to select I accept the terms of the License Agreement and click Next 7 Choose the folder directory in which you would like to install PEAKS The default location is simply C PEAKS 5 To change this location press the Choose button to browse your system and make a selection or type a folder name in the textbox Click Next 8 Choose where you would like to place icons for PEAKS 5 The default will put these icons in the programs section of your start menu A common user preference is on the desktop Click Next 9 Review the choices you have made You can click Previous if you would like to make any changes or click Next if those choices are correct 10 PEAKS 5 will now install on your system You may cancel at any time by pressing the Cancel button in the lower left corner 11 When the installation is complete click Done The PEAKS 5 menu screen should still be open You may view movies and materials from here To access this menu at a future date simply insert the disc in your CD ROM drive 2 4 Registering PEAKS The first ttme PEAKS is run you will be told that the product is not registered Press the OK button and a dialogue will appear Follow the onscreen instructions depending on your requirements For users e
49. will appear as a customized PTM in the set PTM library A listing of all possible built in and custom entered post translational modifications that PEAKS can use as a part of its analysis Residue as used in this manual a residue refers to what remains of an amino acid once it has become part of a peptide or peptide fragment In this manual residues are referred to by their original amino acid names Resolution refers to the resolving power of an instrument On a spectrum this is reflected by how close together two peaks can be and still be resolved Variable modification selecting a post translational modification as a variable modification tells PEAKS that this modification may or may not be applied to the residue s on which the PTM can occur x ions a C terminal fragment holding at least one charge The x ion s mass will be the sum of the masses of the C terminal group plus the intervening neutral amino acid residues plus the mass of carbon monoxide y ions a C terminal fragment holding at least one charge The y ion s mass will be the sum of the masses of the C terminal group plus the intervening neutral amino acid residues plus the mass of 2 H z ions a C terminal fragment holding at least one charge The z ion s mass will be the sum of the masses of the N terminal group plus the intervening neutral amino acid residues after subtracting the mass of ammonia 18 2 Toolbars Main Window Toolbar m New File Th
50. 0 777 4 Spectrum 5 99 0 0 14 820 89 2 0 0 0 0065 3 9455 OrbiSample new 1 428 0 787 m PEAKS 5 presents a list of proteins ranked in descending order from highest score on downward Clicking on any protein will display the peptides matched to that protein in the bottom pane In this case is Serotransferrin precursor from bovine This protein has two matching peptides which you can see in the Peptide List The entire sequence of the protein is shown and the matched parts are highlighted in blue In this case the total matched part accounts for 3 69 of the protein Note that PEAKS 5 groups together homologous proteins which have the same peptide hits Click on the Chart view tab to see charts of the protein peptide score distribution the false positive rate and the decoy database search results The following window will appear 21 Protein Clusters Info Protein Score Distribution Chart Peptide Score Distribution Chart 85 80 i 75 Two detection clusters 70 1 Multi detection clusters il 85 Single detection clusters 2 Peptide score threshold 20 Calculate a an O an to Chart View Protein View Peptide View Denovo View Peptide Score w o o o a 2 o ao Protein Cluster Index Peptide Index False Positive Rate Chart Decoy Search Result Score Threhold Forward Peptide Mat Reverse Peptide Mat False Positive R
51. 00 1400 1500 1600 1700 The Peptide View window summarizes the results for each MS MS spectrum All peptides that match to each spectrum are displayed By default the spectra are listed by ID in the ID column with the corresponding peptide sequence in the Sequence column beside In certain cases one peptide can correspond to more than one spectrum These spectra are then listed in the ID column under a heading entitled Hit Click on to expand the view to see all of the spectra that can be matched by the same peptide The table below describes the contents of the columns in the Peptide View Window A unique identifier for the MS MS spectrum This differs from a scan number since we may have merged several scans together 55 PTMs are listed in square brackets PEAKS probability score m z The measured mass charge value in Daltons for the peptide zo The calculated charge value for the peptide Mr Calc The sum of the theoretical mass of the residues that form the identified peptide sequence from the database Mass 1n Daltons Mass ppm The name of the file RT Retention time elution time for the peptide as recorded in the scan header Scan The scan number Quality A value from 0 to 1 estimated from the spectrum to refer to spectrum quality Attributes like signal to noise total intensity and spectrum tagging are used Mode that the scan step was perfor
52. 1 2 16037993 0 0061 3 8056 OrbiSample m 0 07 2 0 788 es Spetrum3 ANELLINYK 9729 oo 507 3 2 1012 5916 0 0062 08791OrbiSample m 0 365 0 781 vs Spedrum3 ANETLINVK 9729 oo 507 3 2 1012 5916 0 0062 6 0879 OrbiSample m 0 365 0781 c Specrum2 TSDAMINS W 21 85 0 0 695 84 2 1389 6523 0 0132 S 4869Orbisamplem 0 314 Q 785 v Peptide Align Peptide View Denovo View Immonium 110 07 is 8804 68h32 66930 65932 70434 D 1069 43 105148 105246 105346 53525 9 95446 936 45 997 44 93844 4978 8 vers T0933 TIO vist 39417 6 hart view Protein view 70 07 1222 53 1204 58 1194 53 1239 61 102 06 1351 63 1333 62 1323 63 1368 66 Ba 04 1466 66 1448 64 1438 66 1483 68 790 14 272 4 am amm omm amem a 38 N S2 P 806 112553 110752 109754 1142 56 N 630 28 61227 61326 61426 31564 5 B1 P 8 D PT a v a m 200 400 600 ann 1000 1200 1400 1600 LUE gee er n n Info Survey Alignment Error Map 00 The results will be displayed in the same format as was seen for Protein ID Recall that the Protein ID search identified spectra 1 3 4 and 5 The PTM finder search also displayed spectra 2 with the addition of deamidation on N 3 7 Run an inChorus Search Performing the search with the same data by different
53. 2 CO2A1 XENTR 82 97 142695 94 Collagen alpha 1 II chain OS2Xenopus tropicalis GN col2a1 PE 2 SV 1 re P02566 MYO4 CAEEL 99 14 64 92 99 15 225124 52 Myosin 4 OS Caenorhabditis elegans GN unc 54 PE 1 SV 1 FO Q9XXK1 ATPA CAEEL 99 14 17 47 99 15 57787 613 ATP synthase subunit alpha mitochondrial OS Caenorhabditis elegans GN H28016 1 PE 1 SV 1 O P46561 ATPB_CAEEL 99 14 26 98 99 15 57526 82 ATP synthase subunit beta mitochondrial OS Caenorhabditis elegans GN atp 2 PE 1 SV 2 IO P10567 MYSP_CAEEL 99 14 61 09 99 15 101949 57 Paramyosin OS Caenorhabditis elegans GN unc 15 PE 1 SV 1 A lt P4 7991 RL6_CAEEL 99 12 24312 72 605 ribosomal protein L6 OS Caenorhabditis elegans GN rpl 6 PE 2 SV 1 o P21933 ATPB_STRDO 87 52 8712 084 ATP synthase subunit beta Fragment OS Streptococcus downei GN atpD PE 3 SV 1 Lg O P46563 ALF2 CAEEL 99 15 38846 258 Fructose bisphosphate aldolase 2 0S Caenorhabditis elegans GN F01F1 12 PE 1 SV 1 ie P50140 CH60_CAEEL 99 12 60101 055 Chaperonin homolog Hsp 60 mitochondrial OS Caenorhabditis elegans GN hsp 60 PE 2 SV 2 l 2 A9MR77 DNAK_SALAR 14 03 79 54 69319 234 Chaperone protein dnaK OS Salmonella arizonae strain ATCC BAA 731 CDC346 86 RSK2980 GN dnak PE 2 SV 1 67615 73 Chaperone protein dnaK OS Caulobacter crescentus GN dnak PE 2 SV 2 P20442 DNAK_CAUCR 59 09 82 7 Similarly in Compare View all peptides in the result of PEAKS 1 or PEAKS 5 are listed in the first column For each peptide the spectrum id m z and score in
54. 43 for more information on how the quality score is generated The following table describes the contents of the columns in the Peptide Candidates Frame ID A unique identifier for the MS MS spectrum This differs from a scan number since we may have merged several scans together Sequence The sequence of the peptide including modifications if present as determined by de novo sequencing TLC Total local confidence the confidence that we have in the peptide sequence It is calculated by adding the positional confidence for each amino acid in the peptide sequence Average local confidence the confidence that we have in the peptide sequence It is calculated by adding the positional confidence for each amino acid in the peptide sequence and dividing by the total number of amino acids The sequences for a particular spectrum ID as sorted by score TLC The measured mass charge value in Daltons for the peptide Calculated using the measured m z and calculated z we use this as the experimental mass of the peptide The name of the file Retention time elution time for the peptide as recorded in the scan header Scan The scan number Quality A value from 0 to 1 estimated from the spectrum to refer to spectrum quality Attributes like signal to noise total intensity and spectrum tagging are used Mode that the scan step was performed in Frag Mode Mode that the fragmentation step was performed in 47
55. 5 DL oe 1 1 2 3 4 Protein Cluster Index Feptide Index False Positive Rate Chart 30 25 20 15 10 5 0 25 50 75 Threshold False Positive Rate 85 inChorus Image Files Select an inChorus results file Right click and select Export Statistics Graph The following window will appear giving you the option of exporting a Protein Score Chart a Peptide Score Chart an inChorus Result Pie Chart or an inChorus Result Venn Diagram amp Export Statistics Graph Specification Statistics Graph Type De novo 4 A Score Chart 5 Peptide Score Chart False Positive Rate Chart O InChorus Result Pie Chart InChorus Result Yenn Diagram Peptide Number Pie Chart Peptide Number Yenn Diagram Protein Number Pie Chart Protein Number Yenn Diagram Options Select Export File Destination Below are examples of the inChorus Result Pie Chart and inChorus Result Venn Diagram respectively InChorus Protein Number Pie Chart InChorus Protein Number Venn Diagram PEAKS XTANDEM PEAKS only 82 MASCOT Examples for the Protein Score Chart and the Peptide Score Chart can be found above in the Protein ID image files section Compare Image Files Select a Compare results file see chapter 12 for more information on comparing results files Right click and select Export Statistics Graph The following window will appear giving you 86 the option o
56. 62 6 0879 0 781 D i D i 4 i a oO The Peptide View of an inChorus report contains the scores received by each search engine involved in the inChorus search A indicates that the search engine did not find that a protein sequence for that particular spectrum Notice that while PEAKS Protein ID found spectra 1 5 X Tandem found 1 5 and OMSSA found 1 2 5 Click on the Protein View tab Accession ID Mass Display InChorus Score Coverage Query matched Marked Description PEAKS XTANDEM OMSSA Db Search Q29443 TRFE_BOVIN 1 777532 1 I 98 43 5 26 6 Serotransferrin v v v P00330 ADH1_YEAST 2 36691 957 61 53 2 31 1 Alcohol dehydrog v KA The Protein View of an inChorus report displays the proteins that were found and indicates by checkmarks whether the different search engines found that protein or not In this case the X Tandem and OMSSA searches did not generate any extra results that PEAKS did not find but helped to confirm that the first protein is good match Click on the Chart View tab 28 Protein Clusters Info Denovo View Multi detection clusters 1 Two detection clusters 0 Single detection clusters 2 Peptide score threshold rotein View Peptide View Peptide Score hart View Protein Score 96 c
57. 72 22 Q 1278 69 126056 1261 66 1262 66 63984 11 5 74 06 556 24 538 23 528 24 573 26 T 1150 64 1132 62 1133 60 113460 575 81 10 3 6 110 07 693 30 675 28 665 29 710 32 H 1049 60 1031 49 1032 55 1033 55 525 29 9 7 136 08 856 36 838 35 828 36 873 39 Y 912 52 89451 895 49 896 40 45676 8 136 08 1019 43 1001 43 991 43 1036 45 Y 749 46 731 45 732 43 73343 37523 7 d 9 44 05 1090 47 1072 47 1062 46 1107 49 A 586 44 56829 569 32 57037 293 70 6 10 72 08 1189 53 1171 51 1161 51 1206 55 V 515 35 497 34 498 33 499 33 258 18 5 11 44 05 1260 56 1242 56 1232 57 1277 59 A 416 29 398 28 399 26 400 26 208 64 4 v 5 RE gt S o ci 3 co amp 2 S ze is 2 g z 4 Eg Bll b le z ZI gt ca x e a m e 7 ag e 78 2 a o gt gt 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 au 1 1 2h ey o D Info Survey Alignment Error Map a 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 46 Peptide Candidates Frame PEAKS displays the peptide sequence candidates at the top of the screen in the Peptide Candidates Frame You can sort the results by clicking on any of the titles of the columns For example to sort the peptide sequence candidates by ID click on ID Note that all of the peptides that have the same ID have the same mass charge retention time and quality score See page
58. 8 Show unimad Mew PTM Max variable PTM per peptide Database Options Select database Mew Database Edit Database O Paste fasta sequences Advanced Options PEAKS uses a hybrid search technique that requires some sequence tags ba help in Ehe search O Run de nove using different parameters than the above C Run de nove using the same parameters as above default validation decoy search vf Next check the X Tandem box and select the name Note that you will need to use the Ctrl button to select multiple search engines Enter the following settings 25 InChorus Search PEAKS Protein ID sequence library Taxonomy ctrl key for multiple selection a Tandem Sample DE S ba Taxonomy supported Archaea E Aeropyrum pernix omssa Methanoceccus jannaschii Other Archaea Bacteria Mascot Actinobacteria M reversed sequences 2 D al i9N Find models with logta lt 1 v i Sequest F Import Result a 3 residue mista 1 Complete modifications Carboxymethy C specify your own 2 Potential modifications Oxidation M Al Oxidation W Ja 22 ea Deamidation N 9 specify your own Deamidation 0 i iti Oxidation W Deamidatian N Deamidation Q IICAT D 2H 8 C Deamidation N et HOS Semi style cleavage yes 3 gg protein cleavage specification 1 9 Cleavage site 2 Semi sty
59. 90 47 1072 47 1062 46 1107 49 n 586 44 568 29 10 72 08 1189 53 1171 51 1161 51 1206 55 Y 515 35 497 34 11 44 05 1260 56 1242 56 1232 57 1277 59 416 29 398 28 12 72 08 1359 66 1341 62 1331 64 1376 66 Y 345 24 Jell 13 72 08 1458 70 1440 20 1430 71 1475 73 Y 246 18 228 17 14 101 11 K 147 11 129 10 e gt e 3 2 S o oo amp A a a E On e m a gt n r gt gt c E e ci 3 gt m to gt gt ot co Y a EM X I e I I gt mn E eT La a a T Zr 2 gt 25 o z or sg 2 T 2 2 Ta zo qp3 2 T 100 200 300 400 500 600 700 800 900 1000 1100 1200 wh 131 2x 2v Info Survey Alignment Error Map D 0 a 100 200 300 400 500 600 700 800 900 1000 1100 1200 The Peptide ID List shows each spectrum for which PEAKS found a matching peptide Since there may be more than one spectrum that matches a peptide these spectra would be listed together under a Hit node Use the and boxes to expand and collapse the node to see the spectra that are listed together With this dataset spectra 4 and 5 can both be found under one hit 20 The Peptide Alignment Panel contains an Ion Table Spectrum View Pane and Error Map as was displayed in the De novo View seen above For more information about these sections please refer to page 55 Click on the Protein View tab on the upper left hand side The following window will appear
60. AKS22 PEAKS 32 zu uu Peptide Number Venn Diagram PEAKS 11 PEAKS 22 PEAKS 32 only eis un only 26 2 PEAKS 22 only 52 3 PEAKS 32 Compare Chart Compare View Protein View penovo View 80 13 Exporting Data Reports and Printing PEAKS 5 allows you to create reports to share with collaborators colleagues and clients The reports are available in HTML or Microsoft Excel xls formats and follow a What you see is what you get philosophy All the information you see on screen in PEAKS 5 will appear in the exported report For this reason it is important that we complete results filtering and toggling columns before exporting a report 13 1 Export Data in mzxml or mgf In order to export your data file in mzxml or mgf right click on the data file that you wish to export F1 Project view EE Mew Project 3 A Sample 1 3 y DENOWC Data Refine W peaks Denovo t PEAKS 2 Peaks Database Search Spider search inChorus Search Export MGF File Export MzXxML file Show Reports b Delete Fraction Click Export MGF File A window will open that will prompt you to enter a name and a location for the file Click Export or Click Export MzXML File The following window will open Exprot MzXML File Enter the start and end RT in the appropriate boxes Then click the Browse button to select a destination to save your file
61. Advanced button will open a new window which will allow you to be more specific with your digest rules Enzyme Mame Digest Rules Cleavage Site Jo fesidues at Ehe end of a peptide PE start of a new peptide 4nd Or residues at the end of a peptide i start of a new peptide And Or residues at the end of a peptide start of a new peptide 4nd Or residues at the end of a peptide start of a new peptide Find peptides that satisfy the above rules at both ends 32 General Options Max missed cleavages determine the most missed cleavages to allow internal to the peptide in a de novo sequence For instance setting this to 2 and Trypsin as the enzyme then PEAKS will return de novo sequences with up to 2 R s or K s internally Preprocess before auto de novo PEAKS has its own built in preprocessor for removing noise centroiding and peak charge recognition from MS MS data Check this box to turn preprocessing on PTM Options PTM options This list tells PEAKS what kind of post translational modifications to include in its analysis Drag the desired PTM into either the Fixed Modification or Variable Modification box If the desired PTM is not in the list first check the Show Unimod box to show additional PTMs To create a new PTM click on the New PTM button The following window will appear A PTM Editing PTM name Mass Monoisotopic PO Neutral loss mass Monoiso
62. BVU 18 48896 16 1 4 03 1 Na H anti E Q5 484 METN_LEGPA 13 37639 16 1 3 81 1 Methionine impo P25033 HEMO HYACE 10 45648 945 1 3 15 1 Hemolin precurs amp m ASPTRO NHAA1 MYCSJ 14 66444 64 1 3 1 3 Na H anti P49046 LEGU CANEN 12 52762 76 1 2 95 1 Legumain precu Q95220 RECN_STRCO 19 59837 258 1 2 62 1 DNA repair prot Q6C7YS TIM54 YARLI 22 63625 293 1 2 45 1 F Mitochondrial im Click on the Sequence Browser tab and note that instead of highlighting areas of homology in blue areas of mutation are highlighted in red Sequence Browser Sequence Comparison ID Sequence SPIDER Mz z MriCalc Delta M Error p File i Peptides so SpectaS5TVFDN 16 0 az a2 Z 1515 706 121 0594 730r a oamp Matched peptides shown in blue red For SPIDER 1 MOVEVGOIVN THGIRGEVEY ESNSDFTDTR FOQPGEVLTVN HOQNHEEGLTYV al LSYRVHKGFH MLEFEGINNI NDVEGQYEGDY LYGERDHEDI ELAENEYYYS 101 DIIGSTVFDN DNOPIGRVIN IFETGANDVW WVVEGEKEYXLI PYIADVVKEI 151 DIENKTIRIT PMEGLLD After finding a homologous peptide in the database SPIDER will decide what is likely a mutation and what is more likely a simple de novo sequencing error resulting from certain combinations of amino acids having exactly the same mass L I N GG AG G etc As such it reconstructs the real sequence from a de novo sequence and its homologue This is highlighted on the Peptide Details frame o
63. Beta gal Ca wo on Sequence Browser Sequence Comparison gt NCBI BLAST search of POO7ZZ EGAL ECOL Link to retrieve entries containing this sequence From NCBI Entrez Chart View Protein View Peptide View Denovo View Accession ID Description P00722 BGAL ECOLI eta galactosidase OS Escherichia coli strain K12 GN lacZ PE 1 Sv22 Peptides List ID Sequence PEAKS Sc RSD Miz Z 117 115 114 112 Mr Calc Delta Ma Error p File RT Scan i Peptides L Spectrum 1 A 2 PLDNDIGVSEATR 99 0 0 0 801 388 2 1600 8176 0 0562 35 0785 iTRAQSam 0 042 i Spectrum4 T 2 LFISR 71 38 0 0 440 756 2 879 52997 0 0325 36 9196 iTRAQSam 0 256 4 Spectrum5 D 2 WENPGVTQLNR 92 26 0 0 786 887 2 1571 7815 0 022 13 9797 iTRAQSam 0 428 4 Spectrum M 2 SGIFR 49 21 0 33 427 727 2 853 46027 0 0208 24 3872iTRAQSam 0 5911 i Spectrum 8 V 2 DEDQPFPAVPK 3 50 99 0 71 815 43 2 1628 8652 0 0198 12 1407 iTRAQSam 0 76 13 Matched peptides shown in blue SPIDER matches shown in red 1 MTMITDSLAV VLORRDWENP GYTOLNELAA HPPFASWRNS EEARTDRPSQ 51 OLRSLNGEWR FAWFPAPEAV PESWLECDLP EADTVVVPSN WOMHGYDAPI 101 YTNVTYPITVY NPPFVPTENP TGCYSLTFNV DESWLOEGOT RIIFDGVNSA 114 15 4 SILAC Walkthrough Stable isotope labeling with amino acids in cell culture SILAC is a method to metabolically label proteins for relative quantitative comparison One cell population is fed amino acids of normal isotop
64. Charge 3 De novo Retention Time Range min v PEAKS Search Label Options SPIDER Search C Labelling occurs at the MS MS level eg iTRAQ Labelling occurs at the M5 level eg ICAT PTM Finder x Quantification Sample Added Mass Residues Labelling Efficiency Add Label Delete Label Quantification parameter options include the following Basic Options Mass Error Tolerance Quantification is based on the feature of a peptide that identifies its origin in the sample mixture For example in a SILAC experiment one feature is unmodified peptides and the other is peptides modified with Label 13C 6 on arginine or lysine For iTRAQ the feature would be reporter ion m z value The mass error tolerance is for pairing up features Upper Bound of Precursor Charge The peptide may present in different charges Upper bound of precursor charge defines the maximum charge of peptides which are used for counting quantity 107 Retention Time Range The retention time range is for pairing up features For 1TRAQ it is optional Labeling Options Labeling occurs at the MS MS level e g ITRAQ It is for quantification based on the relative intensities of fragment peaks at fixed m z values within an MS MS spectrum Labeling occurs at the M level e g ICAT Itis for quantification based on the relative intensities of extracted ion chromatograms XICs for precursors within a single data set Label f
65. Da Retention time between and min Her e We will use a Quality value greater than 0 65 suggest 0 55 quality filter to remove data with a reprocess Options 2 quality value lower Preprocess MS MS scans CO no already done yes C no than 0 65 As all of the data in this data set is of good quality data we will not remove any data Tasks Running Info Properties o Selection Details OrbiSample mzXIL Total MSI Spectra Total MS MS Spectra Ion Source Fragmentation Made MS Scan Made MS MS Scan Made MS Scan Centroid MS MS Scan Centroid Group MS MS Scan Carter refine Charge Options Filter Quality Process 4 6 ESifnano spray CID CAD IRMPD y and b ions FT ICR Orhitrap Linear Ion Trap False False 6 i 4 55 true 16 using this filter After running data refine there will be new information listed in the Properties file 3 4 Run De novo Sequencing 1 Click the De novo sequencing toolbar icon da Or Select De novo from the Tools menu 2 Enter the settings as shown De nova Save Parameter orbisample Mass Options Data Refine Ace Parent Mass Error Tolerance D 1 De novo Fragment Mass Error Tolerance os PEAKS Search Enzyme Options Enzyme Trypsin ae Mew Enzyme Digest Rule RK SPIDER Search PTM Finder Quantification FTM Gptions me ae es OP Add Fixed gt CQ Fixed Modification 4 Todoacetic aci
66. ENSEE FOR THE SOFTWARE THE LIMITATIONS OF THIS SECTION SHALL APPLY WHETHER OR NOT THE ALLEGED BREACH OR DEFAULT IS A BREACH OF A FUNDAMENTAL CONDITION OR TERM 6 Termination This Agreement is effective until terminated This Agreement will terminate immediately without notice if you fail to comply with any provision of this Agreement Upon termination you must destroy all copies of the Software Provisions 2 5 6 7 and 10 shall survive any termination of this Agreement 7 Export Controls The Software is subject at all times to all applicable export control laws and regulations in force from time to time You agree to comply strictly with all such laws and regulations and acknowledge that you have the responsibility to obtain all necessary licenses to export re export or import as may be required 8 Assignment Customer may assign Customer s rights under this Agreement to another party if the other party agrees to accept the terms of this Agreement and Customer either transfer all copies of the Program and the Documentation whether in printed or machine readable form including the original to the other party or Customer destroy any copies not transferred Before such a transfer Customer must deliver a hard copy of this Agreement to the recipient 9 Maintenance and Support BSI will provide technical support for a period of thirty 30 days from the date the Software is shipped to Licensee Further maintenance and support is available to
67. ERVER The socket used by the 4700 SERVER this is the port that the Oracle database listens to the default 1s 1521 Username to access the Oracle database most likely we do not need to change this the default is tsquared Password to access the Oracle database mostly likely you do not need to change this one either Data extraction procedure 1 Load Spot Set List from the database Do it via menu File Load Spot Set List The extractor will export the peak list of a spot set into a PKL file 2 Open a Spot Set menu File Open Spot Set Spot Set Chooser will help the user to choose a spot set After selecting a spot set click OK to open it The job run information of a spot set will be shown 3 Select a job run There is a button to select before each job run Only the MS MS job run can be selected for export as the precursor information is needed Select a job run and click Convert to do the extraction 34 4 Choose a filename to save After clicking the Convert button the user needs to input a file name and the peak lists of the selected job run will be exported 4 3 Load a New File After making sure that you have the appropriate third party software use the instructions below to load the data files 1 Select New File from the file menu or use the blue file menu icon mi First select the mass spectrometry vendor from the drop down menu or keep the default General setting 2 Select your instrument
68. FSGPAPR 20 41 916 44025 u Spectrum 8 GGLDVR 15 46 0 33 616 32 1 615 33405 0 0213 34 6186 SILACSample mz 0 1415 Spectrum 8 GGIDYR 15 46 0 33 616 32 1 615 33405 0 0213 34 6186 SILACSample mz 0 1415 Spectrum 9 QFAADHALR 36 48 0 78 514 76 2 1027 5198 0 0143 13 89995ILACSample mz 0 1818 Spectrum 10 GGGEGTFR 5 8 D 88 780 35 1 779 35626 0 0135 17 3862 SILACSample mz 0 19 19 2 Spectrum 13 AIR 7 9 0 0 359 21 1 358 23285 0 0301 84 0888 5ILACSample mz 0 2524 I A c Peptide Align Peptide Details 3D View Intensity 1250000 200000 1150000 1400000 150000 Home Scan From 1M To 26 Mz From 479 6 15 3 ITRAQ Walkthrough Isobaric tagging for relative and absolute quantification 1TRAQJ uses isotopic labeling to enable relative quantitative comparisons Up to eight different proteomic samples can be labeled using eight different 1sobaric tags I Creating a Project Click on the Create new project icon or select the New project from the File menu The following window will appear To 483 6 4l Intensity From 32 204834 A New Project Steps 1 Project Properties 2 Project Name Project Location Project Folder To 253991 39 Apply Auto Project Properties iTRAQ Sample D derbyServeriserverDB The 3D view contains 3 axes intensity m z ratio and scan number
69. FTP client software to download the database Open your Internet Explorer and paste the URL into the address bar A file download window will open Click Save 5 Once the database is downloaded you need to make sure that you decompress the file if it 1s compressed using a program such as WinZip or WinRar to extract its contents The file inside the compressed file will be a FASTA format text file a fas or a fasta file 6 Finally put the database file into a directory that PEAKS can access 7 Click Browse to tell PEAKS where the database file is located 8 If the database that you have selected is an EST database check the box labeled EST Database If not leave it blank 100 9 Since you have already selected a FASTA Format Database in Step 2 the Accession number information and the parsing rules for the database headers are shown in the textboxes below in the Advanced Options Fasta Title Format panel Advanced Options Fasta Title Format Rule to parse accessionjid From FASTA title Wait ias Rule Eo parse description From FASTA title eret Accession id URL http f www ncbi nlm nih gov entrez viewer Fcgi db probein amp val lt Accession ID gt Po If you chose an Other in step 2 you must enter parsing parameters yourself by typing in the textboxes Alternatively if our database format is the same as one of the public databases you can choose to apply that database s forma
70. IL AC WaIKtlEOUPT indien erac idera arae aE i deed avr etn eai e 115 I Creatine projecL os dici neo d cate Ee eb fi eve ee e ERE edet fi ere een edad 115 2 Running Protein Identification ecce ecce eee eee e eee eee eese eee e aus 117 3 Running Protein Identification eee e ecce eee eee e eere eee eee eee e eaae 117 A Running Quantification eo eeo eue ee pea oa ra Poo aee e ao Y Fuer aee eaa a tere aee RENT aeo 119 15 3 Label Free Quantification Available in future 120 DENOVO qanm NI LI MEI I UNE NA UI E 121 SPIDER 6 5250 0 0 0 Aoc Orsi LI UT D DII DM DEO DII SD duces Gp c LIS Ee OcUE 121 COUMADIN CATION PRI IEP T A T E m 121 IIS Silber 122 18 1 Terminology and Abbreviations Glossary ecce eere eene enun 122 152 EILEEN TESTE O EEan 123 Main Window Toolbar 4 eicit oie eoo a aa a Nada 123 PEOIGCE V TOW 2 oec nee eepsct eee ivo e ee LO ec iE EE LEES ASTE OO EE TELE EE IEEE EASTER EPIRI 125 Main Processing Window Toolbar ecce eee e eee ee e eere ee eee ee eee eene 125 ESD Mass SCSITVIIPICU MOTO TT 126 Advanced Opl llsns uini tn lr D atin FEE pn t Ca QU 127 19 About Bioinformatics Solutions Inc eee e eee ee eee ee eee ee ee eee eeeeeoeee 128 20 PEAKS Software License eee ee eee e eee eee ee
71. Iti INMCHORLIS Compare Results Perform Filtering Export PepsML Export Excel Export Statistics arap Hide Result De novo Image Files Select a De novo results file Right click and select Export Statistics Graph The following window will appear Export Statistics Graph Specification Statistics Graph Type De novo A 4 Score Chart Options Fle Format Width Height Select Export File Destination The De novo A A Score Chart option will be A A Score Chart selected in the Statistics Graph Type panel Select a file format and height width for your chart and browse your computer to select a destination Beside is an example of the output Number of Peptide D 45 0 50 0 55 0 60 0 65 D 7 A A Score 84 Protein ID Image Files Select a Protein ID results file Right click and select Export Statistics Graph The following window will appear giving you the option of exporting a Protein Score Chart a Peptide Score Chart or a False Positive Rate Chart Export Statistics Graph Specification Statistics Graph Type 5 Peptide Score Chart O False Positive Rate Chart Options Select Export File Destination Below are examples of the Protein Score Chart the Peptide Score Chart and the False Positive Rate Chart respectively Protein Score Distribution Chart Peptide Score Distribution Chart 100 100 B M g 75 75 5 5 a0 oo 5n m c E 25 E 2
72. L We ar it tina S 5 8 i E lth di bE iar Ipe i ley LEE ih i EE m TEN mI i a N Wam CAS mus 3 L9 A3 HLE CE im ji ul sae imr m pm c EA LA node re E imm IE F I IE I T Pal I Im EE Gee j ae ia 3 iE LIE i FTT 1234 ig 1 i2 35 ri 17 1213 48 Lh ua HA iz ia a p ie E 2255 dd HAE nd ae tb Al 1528 98 bist 588 3 Fi i IHA rmx te ia ta E EE LET TE ino in i HE EE a 1m is I i kx MES TEE ETE D i xt run da fal sa ET r Ima nil m tx n DIE 8 p i Di ur a lL i TH TZL 4I Him nim ucl it Lihat Litt m tis iia 1 4 VET J ihe 4 wm iN E Ha a ii sa Dn um 5g 1 57 pm n i JEST j HE su c mx ZH i i 725 MAA Hia TY 42s 28 ra Um fram uh EF P poe a ws L Hii al idc mmi TiN j n aa pa zm zum iw EXT za Er TY zm Iu m UR EI E IA Hi id iu eh 3 Bi Du a wa t 4 1 1 u i PL p ita 290 Aud Ax 2nd 0 ri EN wa 1000 15d Lit Vox 1420 2nd Ted o e nm R rr ed niai pa en ae ey ee ee dre el nmm b irn Lm iac mm aO on en mu Bon EE Longo Tim Lm 13300 pami Ln Leo Note that all spectra can now be identified by the SPIDER search Spectrum 6 is well identified Clicking on the Peptide details tab will di
73. Location of Saved Projects ccce eere eee e ee eere eee ne 39 4 7 Orientns YOUESEIE s aoi eret e eode E a toes 39 Project View Panel ineeeeeeestviteet te eee oe op eesni baee rae ver esee ob oe veve o Passa i bee erae Sa eee gov 39 Properties Panel ese eise over ee tae eso equa eb te even eee N 39 Raw Spectrum Vie Wesss narea 40 SM Data refinement sc cecedecasesscacdedeccsee deck sacuctaceswesedssactebacdesbiecsonccccceseetcedeceeteteccesececuces 42 A MBIBBPICBNOUI SS TT TI TTL TT 42 5 2 Data Refinement Parameters cccccccsccsccscccccccccccccccccccccccccccccccccccccccccccsccccccces 43 IVC RO me SCAMS ENTIS 43 Precursor Charge Correction essei nii aaaeeeaa a aN 43 Falterime MS MS SCAN osinaren a EA EEEa A EEA 43 Preprocessing MS MS Scane scccccccssssssscccccccscccsssssssssssscsssssssssscccccssssssees 43 5 9 Data Preprocessing RESUMES cicccscsscesscitedsccctesseuseseceshaccvsnsusteedseceastabesscscasseccbensseseseass 43 6 De NOVO Sequenclilge aioseiee baee o eee esee epe e ku evv de ore eoa eer a eee Roe eiae ehe e Pra a Ea oea 44 6 1 Setting up Auto De novo Sequencing Parameters ccce eee e eee e eere eee eene 44 Mass OC PULONIS eL 45 Enzyme OptUOHS 55 iii eto YES EE PE PIEEEE E NEEESE armani ELI RETE LEE EI 45 PIM ODLOHS 2 rS E a EIE DERI Is 45 General OpHols cir iot ia nn e e ERI E ia nce eei Eee recie 45 6 2 De
74. Properties Orbisample Ci Peaks Studio 5 0 derbyServerYserverDB Peaks Studio 5 0 derbyServer serverDB Orbisample Project Mame Project Location Project Folder Notes Description Type and organization of project Basic Project Several non labelled samples For comparison each sample can be Fractionated Sample Properties Give this sample a name Select Files Add a File Far this sample i Remove file Fram list Clear list Sample Notes Description Add another sample Remove current sample 13 Enter a name for your project Click the Next button By default the first sample will be named Sample 1 Click the Add a file for this sample button Select the OrbiSample mzxml file from your PEAKS program folder For example CAPEAKS Studio 5 0 Data i New Project Steps Sample Properties 1 Project Properties Give this sample a name Sample 1 v zZ Sample Properties Select Files J Mame Size Date Modified Type ao atajarbiSample mz mML 370 KB 01 21 2009 17 06 05 PM mexml Click Next amp New Pro ject Steps Instrument Details Project Properties Instruments s used to acquire the data Sample Properties du Ep menE betae Instrument Details FT Erap Ton Source ESiinano spray MS Scan FT ICR Orbitrap Fragmentation Type CID CAD IRMPD iy and b ions FT trap etd 5n Scan L
75. S HU 64 117826 305 31 33 0 94 19 83 0 1 F Inversin Inversion of e 8 Q8N6N7 ACBD7_t 66 9790 278 15 6 15 91 1 09 0 1 T Acyl CoA binding domai amp Q96EU6 CF153 F 67 29823 105 _ i 13 72 4 63 10 49 0 1 UPF0399 protein C6orf PEU P13497 BMP1 HU 68 111248 77 11 87 0 81 0 49 0 1 Bone morphogenetic pr N ig Q7Z460 CLAP1_H 69 169449 78 10 32 0 65 1 F CLIP associating protei 5 i QSNSZO AADAT t 70 47351 652 tl 9 85 3 29 1 E Kynurenine alpha amin iB i Q96EP1 CHFR HL 71 73386 586 8 33 0 9 1 F E3 ubiquitin protein liga imet rv37c2 cnlci MO wit 7 757701n 7 o not n co n 1 r1 Eie O SCI M O Dab i 119 There are four peptide features are identified and calculated for quantification of Human Filamin A Accession ID Description Peptides List ID Sequence PEAKS Score M Z Z Mr Calc Delta Mass Error ppm RSD File RT Scan Quality Peptides Spectrum 12 YGGQPVPNFPSK 3 99 0 648 84 2 1295 6604 0 0051 3 957 0 92 SILACSample 0 28 27 0 785 Spectrum 13 DVDIIDHHDNTYTVK 3 67 65 895 94 2 1789 8579 0 0076 4 2285 0 87 SILACSample 0 2928 0 789 Spectrum 19 TGVELGK 3 PTHFTVNAK 3 99 0 855 99 2 1709 9502 0 0153 8 9234 SILACSample 0 39 38 0 789 Spectrum 27 DVDIIDHHDNTYTVk 46 55 595 62 3 1783 8376 5 0E 4 0 2737 0 93 SILACSample 0 8871 0 787 Spectrum 35 VANPSGNLTETYVQDR 99 0 882 44 2 1762 8486 0 0168 9 5559 0 25 SILACSample 1 0787 0 789 Spectrum 44 VEPGLGADNS
76. VVR 99 0 1312 6899 1 1311 6782 0 0044 3 3503 0 54 SILACSample 1 22102 0 813 PEAKS provides the 3D view of each peptide feature for visual validation For example spectrum 12 is among the feature of YGGQPVPNFPSK Input the scan range of 25 28 and m z range of 644 652 Click the button of Apply The 3D view of the feature is displayed x D iea TT F is xm oca num 15 3 Label Free Quantification Available in future Label Free quantification relies on the changes in analyte signals directly reflecting their concentrations in one sample relative to another This technology employs overall spectral intensity normalization by interpreting signals of molecules that do not change concentration from sample to sample By comparing two or more spectra PEAKS can determine the constant intensity ratio between the unchanging analytes forms the basis for identifying the non changing concentrations making spiking unnecessary 120 16 References De novo Bin Ma Kaizhong Zhang Christopher Hendrie Chengzhi Liang Ming Li Amanda Doherty Kirby Gilles Lajoie PEAKS Powerful Software for Peptide De novo Sequencing by MS MS Rapid Communications in Mass Spectrometry 17 20 2337 2342 2003 SPIDER Y Han B Ma and K Zhang SPIDER Software for Protein Identification from Sequence Tags Containing De novo Sequencing Error Journal of Bioinformatics and Computational Biology 3 3 697 716 2005 Quantification Yang W Ch
77. Window OrbiSample mzxML x a TIC p wv 4 f z un r3 v Q3 o c 200 400 600 800 1000 1200 1400 1600 Lug J n CURA FAT OrbiSample mzxML ms 1 RT 0 01 scan 1 TIC 1 27E8 802 91 0 o LJ ew e 200 400 600 800 1000 1200 1400 1600 RT 0 01 1 5 min 3 yl l til 2X 2 OrbiSample mzXML ms 2 mz 802 91 2 0 RT 0 07 scan 2 TIC 1 79E7 15 The information that is displayed by default pertains to the precursor scan To the left of the window is the Total Ion Current TIC The graph in the upper right corner displays a survey scan with its corresponding tandem scans below Click on the MS MS tab to see the graphs that were generated from the tandem scan For more information on the functions and tools found in these windows see page 40 3 3 Perform Data Refinement 1 Click the Data Refine toolbar icon s Or Select Data Refine from the Tools menu Data Refine 2 Enter the settings Tools Data Refine as shown Merge Options Y Data Refine Merge scans of the same peptide yes no De novo Retention time window For raw Files only min For more details on m z tolerance av PEAKS Search is gt setting up the _ SPIDER Search Charge Options parameters for data Correct precursor charges G yes O no s _PTM Finder refinement refer to Minimum charge 1 Maximum charge 4 Quantification page 43 P Filter Options Filter MS MS scans G yes O no Precursor mass between and
78. al uU iF Li B i i BIOUINPORMATICS SOLUTIONS PNG PEAKS Studio 5 User s Manual Bioinformatics Solutions Inc 470 Weber St N Suite 204 Waterloo Ontario Canada N2L 6 2 Phone 519 885 8288 Fax 519 885 9075 Please contact BSI for questions Of suggestions for improvement ELS Mim ESTRENO m 6 L2 New Features in PEAKS S uice eieut ebenso aes sa tabao eed eru E ei oe bid Fes usa eese oae Coco aaia 6 LS WoOrkOW me pm 7 1 4 Guidelines for Using this Manual eee e ecce eee eee e eee e ee eee esee ee ssssasne 7 L5 SCODE denen ve dp D Pa ID nisi ui 7 1 6 Service and SuUDDOEL ui NET PERI Ee IE OE DI ione e NOE EE REOS EU CR DIS Ines Uo NOH Eee RN ES 7 2 Getting Started with PEAKS 5 ote ie e PEE OI Fi Ee SENI E e ceu eoe FERES EE Po cU eu Seele 8 Del WACK ACE C Onten S ooo ice E OE Oi iiM DH 8 2 2 System Requirements oett Eoo ea eese iv toris NE E estet st EE 8 2 3 Installation for Windows Users cccccccscccsscccsscccccccccccccccccccceccccecccsccccccccsccccccceees 8 ZA Resistens PEAKS e M 10 2 9 DEUUP PEAISS PEOIGEOBICOS seircean Ea EEEE aas 11 20 Set p PEAKS COMMPUT AON cecdcsiseivastesccedsscedevssissdendostevecasbesicedewecevseassieoteecoerseiees 11 3 Quick Walkthr Daenen eenen R NEEE E NEEE ES 12 JT Create a saniple Data Was siwciasaveascseveciasasecesdesssseacarsaveassnscecacasecsneraesdessiusereusenacases 12 Deze Create
79. alculator from the Tools menu The following window will appear PEAKS File Help Input Sequence or Mass Da O b ion y ion 9 non specific PO Peptide Mass Charge Miz Mass Margin of Error prediction of sequence Sequences Mass Dat In order to use the mass calculator input the peptide sequence or enter the mass in Daltons Indicate if the sequence contains b or y ions or if your search is non specific For example select b 10n input the sequence ACDR and click the Calculate button You should see the following Input Sequence or Mass Da 9 b ion 2 y ion C non specific Peptide Mass Charge Miz Mass You can change the charge and use the arrow on the right to calculate the precursor mass or use the arrow on the left to calculate the peptide mass Or for another example change the margin of error to 1 Da input a mass of 146 Da and click the Calculate button In the sequences box you should see the following predicted sequences Sequences Mass Dal 5 144 0535 Sg i4 053s 126 Use the Clear button to start again Advanced Options Clicking on the Advanced button will display the following Advanced Options eJicJloJlesiFJisJ iJ it ie Le im Jin jt Jti Jb Ji Jn b JO Jos Modification Mass Residues Include Acetylation M te 42 010567 ADTEQGILM Acetylation E 42 010567 K Anywhere Amidation 0994016 ARNDCEQG
80. appear Mass Options Parent Mass Error Tolerance Fragment Mass Error Tolerance Enzyme Options Enzyme Digest Rule RI Fiat Find peptides thak satishy af botn ends PTM Options Mame Mono Residu Acetylation N term 42 010567 ADCEQ Acetylation E 42 010567 K Amidation 0 984016 mc Applied Biosystems original ICAT TIM do 442 225 C Applied Biosystems original ICAT TM d 450 2752 C Applied Biosystems cleavable ICAT TIM light 22712698 C Applied Biosystems cleavable ICATITM heavy 236 15718 C Shaw unimad Mew PTM Max variable PTM per peptide 3 General Options a Preprocess this data on the fly fdeconvolute filter noise centroid Report up to peptides 5 z 3 To change any of the following parameters now is the time Mass Options Parent mass error tolerance Determine how much random and systematic experimental error on the parent precursor ion PEAKS will allow for in its analysis As you have previously selected your instrument PEAKS will provide the suggested error tolerances Type a tolerance in the textbox and choose units from the dropdown list Using PPM allows for larger errors at larger m z values PEAKS will be very stringent concerning this value so new PEAKS users should try setting this a little higher than past experience may suggest if sensitivity is a concern Fragment mass error tolerance Determine how much random and systematic experimental
81. ar on the list or 1s different than what 1s listed you can select the New PTM button and the PTM Editing window will open Fill in the information pertaining to the PTM of interest For a more in depth explanation of creating a new PTM see page 53 To select a PTM as Fixed or Variable drag the PTM into the Fixed Modification or Variable Modification box If you drag over an incorrect PTM simply drag it back to the PTM Options list Note that in previous versions of PEAKS only fixed PTMs were allowed however PEAKS version 5 0 allows variable PTMs as well when using the new block search Max variable PTMs To reduce uncertainty PEAKS de novo sequencing vocabulary can be limited by restricting the number of variable PTM found on a peptide Specify a number by typing it into the box To lift such restrictions type a very large number longer than the length of the peptide Filter Options As the SPIDER search is computationally intensive it is not recommended that you run all of your de novo sequencing peptides against the database only those that cannot be well explained De novo score A A threshold The SPIDER search requires a good sequence tag from de novo to be able to find good quality homologous proteins Enter a value for the de novo score threshold The recommended threshold is 0 5 Peptide score threshold Because there is no need to run SPIDER on peptides that already were found to have a good match duri
82. arger m z values PEAKS will be very stringent concerning this value so new PEAKS users should try setting this a little higher than past experience may suggest if sensitivity is a concern Fragment mass error tolerance Determine how much random and systematic experimental error on the fragment daughter ion PEAKS will allow for in its analysis Type a tolerance in the textbox Again new PEAKS users should try setting this a little higher than past experience may suggest Precursor mass search type If the precursor mass 1s monoisotopic value check monoisotopic Check average otherwise Enzyme Options Enzyme Indicate which type of enzyme was used to digest the sample Choose from a dropdown list of enzymes Note that you cannot delete or change the details of a built in enzyme and therefore the Delete enzyme button and the Digest Rules panel will be grayed out If your enzyme or combination of enzymes is not in the list click the New Enzymes button You will then be able to enter a name for your enzyme digest rules see below and select if you would like to find proteins that satisfy the rules at both ends This option is grayed out for built in enzymes Digest Rules This is how you specify where your enzyme will cleave the protein between two amino acids to create peptides The letter X denotes any amino acid in this position while set brackets indicate any amino acid except the one in the brackets Clicking on the
83. ate False Positive Rate 96 jOoOjojojojojojojojojojojojololololojojo m ojojojojojojojojojojojojojojojojojojo 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 B 3 3 3 50 60 70 Threshold The graphs display protein and peptide scores as well as information on the false positive rate which is generated from the decoy database search Please see page 61 for more information about the chart view 3 6 Run PTM Finder Using the PTM finder you can identify any additional PTMs and increase the coverage of the proteins that we have found It is important to note that the PTM Finder can only be applied to a Protein ID results file As it is very time consuming to run Protein ID with many PTMs this allows searching for more PTMs in less time Make sure that you click on a Protein ID result before performing a PTM Finder search 1 Click the PTM Finder toolbar icon R Or Select PTM Finder from the Tools menu 2 Enter the settings as shown 22 A PTM Finder PTM Finder m orbisample Mass Options General Options Data Refine Parent Mass Error Tolerance Preprocess this data on the Fly Fragment Mass Error Tolerance Max Missed Cleavages 15 De nova l l 7 EN Enzyme Options Digest Rule Delete Enzyme PEAKS Search SPIDER Search x PTM Finder Eins eha Advance Quantification PTM Options
84. cate an equal mass substitution common non critical de novo errors lt these brackets gt indicate an equal mass substitution and a mutation Matched peptides shown in blue SPIDER matches shown in red ec 51 SGPFVSCVERK TSHMDCIKAI SNNEADAVTL DGGLVYEAGL KPNNLEPYVA 101 EFHGTKDNPO THYYAVAVVK KDTDFKLNEL RGKKSCHTGL GRSAGWNIPM 66 When simply identifying exact peptides from the database using PEAKS Protein ID or inChorus there s no need to reconstruct the real sequence Clicking on Protein View will again yield a similar display as was seen for PEAKS Protein ID see page 58 however where there were blue regions to indicate areas of homology when performing a protein ID search there are now red regions to indicate areas of mutation SamplData_draft mzXML x DENOVO 1 05 Dec 08 14 38 x PEAKS 1 05 Dec 08 14 39 x PEAKS 2 05 Dec 08 14 44 x SPIDER 3 05 Dec 08 15 06 x SPIDER Score Coverage Query matched Marked Description Accession ID Mass Display 9 DB Search H Q29443 TRFE_BOVIN 1 77753 20 1 5 26 4 Serotransferrin 5 Q4LSU1 RIMM STAHJ 21 19309 496 1 7 78 1 Probable 165 rR B TW44 GIDB FRAP2 9 23253 205 1 6 37 1 F Methyltransfera r Q8AA41 ISPE BACTN 11 30533 79 7 1 4 74 1 C 4 diphosphocyti f P19231 32K_BNYYG 28 31869 223 1 1 4 26 1 4 RNA 4 uncharac a Q15N42 NHAA2 PSEA6 17 42666 598 1 4 24 1 Na H anti F Q8D666 NHAA2 VI
85. containing the words Human human Rat or rat 76 So type in the regex KeratinITrypsin humanlrat and press the Enter key If PEAKS confirms that this is a valid regular expression it will put a check in the Valid Java Regex box 3 Goal Setting a protein mass range If we know the approximate mass of the proteins you are interested in you can eliminate all proteins that are not close in mass Add two filters Protein Filters Mass gt 12000 and Protein Filters Mass lt 32000 TI 12 Complex Analysis 12 1 Creating a project for complex system PEAKS 5 is able to analysis MS data from very complex systems The data analysis scheme is organized as follows 42 Project nodes i Sample nodes File nodes Below is an example of a project that contains three samples that were generated using the following fragmentation methods ETD CID and CID ETD Each sample has three files i PEAKS File Tools Window Help ig 3 UG FN Proj ED CID_OL RAW CID 02 RAW E CID 03 RAW GJ ETD 0 4 ETD Di RAW ETD 2 RAW 0 9 ETD O3 RAW Jl CID ETD f CID ETD Di RAW E CID ETD 0z RAW sf CID ETD D3 RAW Tasks Running Infa Properties Selection Details thermo data Project Mame Total Samples thermo data a 12 2 Integrating data analysis Within a project the data is analyzed either file by file PN Project view or sample by sample B
86. d derivative IHamaserine 29 9928 IHomoserine lac 48 0034 IHydraxylatian 15 9949 PKDNR Y E en SP LK JON add variable gt e Variable Modification Todoacetic acid 58 0055 c v Show unimod Mew PTM Max variable PTM per peptide General Options Preprocess this data on the fly deconvolute filter noise centroid Report up to f peptides Note that we are not going to preprocess this data on the fly as we have already preprocessed the data during the data refinement stage We will also choose to report only one peptide per spectrum for simplicity s sake You can save the parameters that you used for future reference by clicking on the Save Parameter button For more information on setting up de novo parameters see page 44 Click OK to commence analysis For this sample it takes just over a minute The PEAKS auto de novo algorithm derives sequence candidates for each of the six spectra in our example data file Take a look at spectra ID 1 Notice that the number in square brackets refers to the modification which in this case 1s 10doacetic acid derivative After de novo sequencing is complete the following will appear in the Main Processing Window 17 Id Sequence TLE ALC Fank miz Z Mass File RT Scans DNPOTH Y TAMANE 93 34 D 57 1 02 91 z 15603 7993 OrbiSample mezxML D ny z ATSDALDMNLK o O59 al 695 2 1389 6523 RIKDINGNFEER ee
87. ders select the top level folder to load them all at once Shimadzu Data Shimadzu mass spectrometer data can be loaded provided that the Shimadzu software is installed on the same computer as PEAKS 5 Applied Biosystems Data WIFF data from Applied Biosystems Sciex QSTAR or QTRAP mass spectrometers can be loaded provided that the Analyst QS Analyst 1 4 1 for QTRAP software and the MSX plug in are installed on the same computer as PEAKS 5 The MSX tool is produced and sold by Infochromics Ltd and 1s available at cost from Bioinformatics Solutions Inc Please contact a BSI sales representative to obtain an evaluation or full license Varian Data A conversion tool is embedded into Varian s data acquisition software which allows the conversion of Varian raw data into pkl files which can be immediately read by PEAKS The trans type data raw is converted in Varian programs by clicking File Save As and selecting the pkl file format or by clicking File right clicking Export and selecting pkl If you are viewing a chromatogram with the Varian software all the spectra data in the viewed chromatogram is converted to the pkl format Likewise if you are viewing a single spectrum and choose to convert the data only the viewed spectra will be converted Waters Micromass MassLynx Data PEAKS 5 can import RAW data from Waters MicroMass QTOF instruments using a utility called wolf exe originally created as part of the Sas
88. des are highlighted in blue letters on the sequence A more intense blue indicates a more confident match The background colors indicate similarity between the sequences A dark background indicates regions where residues or nucleotides are identical in all sequences a light background indicates similarity across some sequences and lowercase letters on white background highlight differences A dash 1s displayed where a gap had to be introduced in one sequence to complete the alignment 60 OrbiSample mzxML x PEAKS 1 30 Jan 09 11 35 x Accession ID DB Search li 029443 TRFE BOVI POOSSO ADHi YEAS QOY4K1 AIMI HUM Sequence Browser Sequence Comparison Chart View Protein View Peptide View Denovo view Open browser Display inline Mass Display PEAKS Score 9 5 Coverage Query matched Marked 1 i es RCRNNENEN 98 33 5 26 4 2 36691 957 I 59 38 2 31 1 3 188674 52 2 96 D 64 1 Description Serotransferrin Alcohol dehydroc Absent in melanc Q29443 TRFE BOVIN POO330 ADH1 YEAST QOY4Kl1 AIM1 HUMAN Q29443 TRFE BOVIN P00330 ADH1 YEAST QOY4K1 AIM1 HUMAN Q29443 TRFE BOVIN PO0O0330 ADH1 YEAST QOYAK1 AIM1 HUMAN Q29443 TRFE BOVIN P O0330 ADHl1 YEAST QOY4K1 AIMi HUMAN Q29443 TRFE BOVIN POO330 ADH1 YEAST QOYAK1 AIM1 HUMAN Q29443 TRFE BOVIN POO330 ADH1 YEAST QOY4Kl1 AIM1 HUMAN Q29443 TRFE BOVIN P00330 ADH1 YEAST QOY4Kl1 AIM1 HUMAN Chart View Cl
89. e Enzyme Details panel Note that you cannot delete or change the details of a built in enzyme and therefore the Delete enzyme button and the Digest Rules panel will be grayed out Create a new enzyme Click on the New Enzyme button Digest Rules This is how you specify where you enzyme will cleave the protein between two amino acids to create peptides The letter X denotes any amino acid in this position while set brackets indicate any amino acid except the one in the brackets You can also choose to select the check the box Select peptides that satisfy the above rules at both ends if you desire The example below shows a combination of Trypsin and Asp N amp Configuration Enzyme Enzyme List PTM Trypsin and Asp N combination o lt Built In gt Arg C al Database lt Built In gt Asp M Delete Enzyme lt Built In gt Asp M N terminal Glu lt Built In gt Chymatrypsin E Parameters Built In gt CNBr lt Built In gt Glu C bicarbonate Built In Glu phosphate nel Enzyme Details Enzyme Name Trypsin and Asp N combination Digest Rules Cleavage Site a fesidues at the end of a peptide KR HP start of a new peptide And Or residues at the end of a peptide Kx E D 3d stark af a new peptide AndjOr residues at the end of a peptide m start of a new peptide And Or residues at the end of a peptide IM l o start of a new
90. e Size Date Modified Type Instrument Details Instruments and Files To associate a File with an instrument drag it From the list above and drop it onto the instrument in the list below Use CTRL Click or SHIFT Click to select several Files c3 LTO FT Ultra Hybrid FT FT etd 4 WSimon datatiain 266 RAW IO LCQ Fleet Ion Trap 4 WMSimonidataliaini297 RAW ILCO Fleet Ion Trap pad 4 Wsimontdataliaini274 RAW 4 5 Open a Project Go to File and select Open Project or Open Recent Project m to access stored projects You can also select Close project rz to close a project that 1s open E window Help Mew Project Ctrl Shift h Mew File alr ShiFE OmenProject CtrheAlbeo A Open Recent Project Close Recent Project b i Save As CEri ShiFE4 5 Oo Exit chro 38 4 6 Changing the Location of Saved Projects Projects are saved in the location that is listed in your Preferences window To modify your preferences select the Preferences toolbar icon or select Preferences from the Window menu Select General on the left hand side of the window The default Output Directory and Project Folder locations are listed in the Default Output Directory panel Please note that the defaults seen here may differ from your default locations depending where you downloaded PEAKS Click on the Browse buttons to change either of these
91. ect But PEAKS doesn t know which instrument 66 1 99 duced them the Instruments and Files Sample Properties CO Mame Size Date Modified Type window will open next Instrument Details onidataljaini297 RAW 84 327 KB 03 03 2006 06 54 00 AM Instruments and Files onidata iain274 RAW 91 301 KB 03 02 2006 11 50 00 AM The top of the window will _ onidataliain 266 RAW 171 136 KB 03 02 2006 10 05 00 AM contain a list of all files selected for the project To associate a File with an instrument drag it From the list above and drop it onto the instrument in the The lower part of the list below Use CTRL Click or SHIFT Click to select several files 1 1 LTQFT Ultra Hybrid FT FT etd window contains the viu in adi instruments selected in the ie ieee nna esta Instrument Details window aT 8 In order to indicate which instrument was used to generate each file drag the file from the list above and drop it onto the instrument in the list below In order to drag and drop multiple files at once use Ctrl Click or SHIFT Click Use the reset button to return the files to the list at the top of the window if you make an error Steps Instruments and Files Project Properties This list contains all the Files you selected For this project But PEAKS doesn t know which instrument produced them Sample Properties 9 Click on the finish button and the file will begin loading Mam
92. efore uploading to the server 5 1 Run Data Refine To begin the refinement of data from a whole MS MS run 1 In the Project View Frame select the data file s containing the data that you wish to refine 2 Click the Data Refine toolbar icon la Or Select Data Refine from the Tools menu A Data Refine Tools X Data Refine Data Refine Merge Options Merge scans of the same peptide Retention time window For raw files only miz tolerance Charge Options Correct precursor charges Minimum charge 1 Maximum charge 4 Filter Options Filter MS MS scans Precursor mass between Retention time between and 200 Quality value greater than suggest 0 55 Preprocess Options Preprocess MS MS scans no already done The Data refinement options window will appear 3 Choose the data refinement tools you wish to use by clicking the yes radio button next to each one See the information below to help you decide on proper refinement parameters 5 2 Data Refinement Parameters Merging Scans In DDA mode a mass spectrometer will often produce several tandem ms MS MS scans of the same peptide To increase the intensity of real signal peaks within these scans and to reduce the size of the whole data set it makes sense to merge MS MS scans of the same peptide together To avoid improper merging of MS MS scans of different peptides we make sure that the measured parent ion masses
93. elect peptides For display sp Q29443 TRFE BOVIN DenovoDNPQTHYYAVAUVEK LLELTELLE ETT T T I Recon DNPQTHYYAVAVVK LLELEEEEL E I HomologDNP QTHY Y AV Av VK Matched peptides shown in blue SPIDER matches shown in red 1 MRPAVRALLA CAVLGLCLAD PERTVRWCTI STHEANKCAS FRENVLRILE 51 SGPFVSCVKK TSHMDCIKAI SNNEADAVTL DGGLVYEAGL KPNNLEPVVA 101 EFHGTK NSO THYYAVAVVE KDTDFKLNEL RGKKSCHTGL GRSAGWNIPM 151 AKLYKELPDP QESIQRAAAN FFSASCVPCA DOSSFPKLCOQ LCAGKGTDKC 2001 ACSNHEPYFG YSGAFKCLME GAGDVAFVKH STVFDNLPNP EDRKNYELLC 251 GDNTRKSVDD YOECYLAMVP SHAVVARTVG GKEDVIWELL NHAQEHFGKD 301 KPDNFOLFOS PHGKDLLFKD SADGFLKIPS KMDFELYLGY EYVTALONLR 351 ESKPPDSSKD ECMVKWCAIG HQERTKCDRW SGFSGGAIEC ETAENTEECI 401 AKIMKGEADA MSLDGGYLYI AGKCGLVPVL AENYKTEGES CKNTPEKGYL 451 AVAVVKTSDA NINWNNLKDK KSCHTAVDRT AGWNIPMGLL YSKINNCKFD 57 At the top of the Peptide Details frame is the accession number of the protein that corresponds to the peptide that you chose in the Peptide View window If more than one protein matches a single peptide you will be able to select these additional proteins using the dropdown menu Below this you will see a simple alignment between the original de novo sequence for this spectrum if available the peptide found in the database and the reconstructed sequence Letters on a green background and with vertical bars indicate agreement Color codes on the de novo sequence letters still indicate positi
94. en W Rogers I Ma B Bendall S Lajoie G Smith D PEAKS Q Software for MS based quantification of stable isotope labeled peptides Bioinformatics Solutions Inc Genome BC Proteomics Centre University of Western Ontario ASMS 2006 poster WP531 18 Appendix 18 1 Terminology and Abbreviations Glossary a ions an N terminal fragment holding at least one charge This is a fragment of the peptide derived from b ions The a ion s mass will be the sum of the masses of the N terminal group plus the intervening neutral amino acid residues subtract the mass of carbon monoxide b ions an N terminal fragment holding at least one charge This is a prefix fragment of the peptide The b ion s mass will be the sum of the masses of the N terminal group plus the intervening neutral amino acid residues BSI Bioinformatics Solutions Inc The makers of PEAKS and other fine bioinformatics software c ions an N terminal fragment holding at least one charge This is a prefix fragment of the peptide The c ion s mass will be the sum of the masses of the N terminal group plus the intervening neutral amino acid residues plus the mass of ammonia Deconvolution rearrangement of the spectrum to show each monoisotopic peak as if it were singly charged Thus to reposition them on the scale PEAKS multiplies the m z of ion s that were doubly charged by two minus the mass of 1 H Note that the deconvolved scale PEAKS shows is at 1 F
95. ensities of fragment peaks at fixed m z values within an MS MS spectrum For example 1TRAQ and Tandem Mass Tags e Label free Label free quantification based on the relative intensities of extracted ion chromatograms XICs for precursors in multiple data sets aligned using mass and elution me All three protocols are fully implemented within PEAKS Q The flow chart is shown below Import Protein PEAKS Analysis Data gt ID gt Quantitation gt Report ks 15 1 Setting up PEAKS Q Parameters 1 In the Project View Frame select a PEAKS Search result file 2 Click the PEAKS quantification toolbar icon Q Or Select Quantification from the Tools menu The following window will open displaying the quantification parameters Quantification Tools Quantification Basic Options Data Refine Mass Error Tolerance Da v Upper Bound of Precursor Charge 3S De nova Retention Time Range min v PEAKS Search Label Options SPIDER Search Labelling occurs at the MS MS level eg TRACE C Labelling occurs at the MS level eg ICAT PTM Finder y Quantification Sample Reporter Ion Da Labelling Efficiency 952 4dd Label Delete Label Slightly different options will be available if you select labeling at the MS level Quantification Tools QuantiFication Basic Options Data Refine Mass Error Tolerance 1 Da Upper Bound of Precursor
96. entraid False prompt you to enter this information MS MS Scan Centroid False 39 Raw Spectrum View Opening the raw file in PEAKS will display Br the following graphs in the Main Processing Window The MS tab is selected by default and represents the precursor scan On the left wil hand side of the screen is the total ion current MMnR M HH CubaeSagpie put enim TIC Depending on how the file was generated there may be simply a list of spectra and not a TIC graph The retention time 1s plotted against the vertical axis Clicking on the TIC graph will move the red line and display the ms spectra to the right of the TIC graph that corresponds to the selected retention time Alternatively use the up and down arrows found on the keyboard to move through the TIC If the ms2 scans is available it will be displayed below the corresponding ms scan Al Lil li n TE Ore ee ee h ee ee To zoom either on the X or Y axes select the 2X or 2Y buttons respectively To scroll in even more click the button on the left of your mouse and drag the arrow to the side To increase the intensity of the peaks use slide the scroll bar on the left hand side up and down Selecting the 1 1 button will bring you back to the original image where the entire spectrum is visible Scrolling over the spectrum will display the m z ratio and the height intensity as a percentage of 100
97. erby Jar Location The Derby Jar Location panel will list the location of the Derby Jar file by default If you would like change this location check the User Define box and click on the Browse button to select a new location Click the Apply button to save any changes you have made Performance Clicking on Performance on the menu on the left hand will open the following window eh Preferences General Performance Lao i B RMI Connections Computer PerFormance ol Derby Database ee 1 ies Performance Single Core Sig Cane TS H Instrument Search Engine Show 3D view Ion Settings Fast Loading Files 90 Computer Performance Select the number of cores that your computer contains 1 e single double or quad core Please note that the setting of number of cores that you are able to use must comply with the license 3D view PEAKS will display a 3D view with your quantification results Check the Show 3D View box to enable this function PEAKS 5 comes with the Java3D program to support the viewing of 3D images Fast loading files This function is for raw file loading If you check the Fast Loading Files box PEAKS will only load spectrum header information without the peak list Please note that if you set up a project which contains more than 20 raw files this function will not work well due to memory issues and you should uncheck the Fast Loading Fil
98. ers list on the left by clicking on it Options for this filter will appear in the Edit Filter frame Once you have set the options that you would like for the filter in the Edit Filter frame drag the filter that you would like into the Selected Filter list on the right hand side Click OK to apply the filter that you have selected to the current file If you would like to add another filter you can repeat the process continuing to add as many filters as necessary In this way it is also possible to have two filters on the same property we can set a range of protein mass for instance by applying one filter on the upper bound of the mass and adding another filter to be the lower bound of the mass We can also have more complex filters that involve multiple properties Score 9 For example let s say that you want to show only proteins with more than Filter peptides based on their one high scoring greater than 60 score peptide a standard requirement 50 for publication Double click on the Peptide Filters folder Select Score Filter Edit the filter to select peptides that have a score that is greater than Options 60 1n the Edit filter frame Equal to Greater than Lesser than Not equal to Selected Filters De Novo Filters i Peptide Filters Now drag Score filter from the Possible Filters frame to the e Score fiter 60 0 Selected Filters frame
99. es box Click the Apply button to save any changes you have made Instrument Preferences This section will allow you to change any preferences for the following instruments ABI Bruker Shimadzu and Varian ABI wiff Clicking on Instrument and then ABI wiff on the menu on the left hand side will open the following window A Preferences E General ABI wiff Instrument i ABI wifF Default wiff raw file convertor location g Bruker yep baf Fid C Program Files InfFachramics MSX MSX exe f Shimadzu AXIMA rur 2 Varian xms Search Engine Raw File convertor options Ion Editor ABI raw Files may contain several samples do you want to merge all the samples into one data ser C yes no C Survey spectrum centroiding Praduct spectrum centroiding Default wiff raw file convertor location Click Browse to tell PEAKS the location of the Default wiff raw file converter 91 Raw file converter options ABI raw files may contain several samples By default these samples are not merged into one data set Select yes if you would like PEAKS to merge all the samples into one data set For PEAKS to work optimally it is important to select if the survey spectrum or the product spectrum has been centroided Click the Apply button to save any changes you have made Bruker yep baf fid Clicking on Instrument and then Bruker yep baf fid in
100. f Peptide View 67 9 PTM Finder 9 1 Setting up PTM Finder Parameters 1 Select a Protein ID results file to perform a PTM finder search on Note that you cannot perform protein ID on a raw file or de novo results 2 Click the PTM icon on the toolbar R Or Select PTM Finder from the Tools menu The PTM Finder Options window will appear A PTM Finder E x Tools PTM Finder Se orbisemple vj Mass Options General Options Data Refine Parent Mass Error Tolerance 0 1 Da V Precursor Mass Search Type _ Preprocess this data on the fly De novo Fragment Mass Error Tolerance 9 8 Da Monoisotopic O Average Max Missed Cleavages if PEAKS Search Peme Apion Enzyme Trypsin v SPIDER Search Digest Rule RK MP Y PTM Finder nd peptides that satisfythe above rule at both ends Quantification PTM Options Name Mono mass Residue site o Fixed Modification 4 hydroxynon 156 115 CHK Homoserine 29 9928 M amp xc Homoserine lac 48 0034 M amp c Hydroxylation 15 9949 PKDNRY ICPL heavy 111 0416 K X amp N ICPL light 105 0215 Kk X amp N Iodoacetic acid 58 0055 C Lipoyl 188 033 K Methyl ester 14 0156 DE xJ c Methylation 14 0156 CKRHDENG O18 label 2 0042 STY X ac Variable Modification Propionamide 71 0371 C Trimethylati
101. f exporting a Peptide Number Pie Chart a Protein Number Pie Chart a Peptide Number Venn Diagram or a Protein Number Venn Diagram amp Export Statistics Graph Specification Statistics Graph Type De nova 4 4 Score Chart Protein Score Chart Peptide Score Chart False Positive Rate Chart InChorus Result Pie Chart InChorus Result Yenn Diagram C Peptide Number Yenn Diagram Protein Number Pie Chart 5 Protein Number Venn Diagram Options Select Export File Destination Below are examples of the Peptide Number Pie Chart the Protein Number Pie Chart the Peptide Number Venn Diagram and the Protein Number Venn Diagram respectively Peptide Number Pie Chart Protein Number Pie Chart PEAKS 11 only 10 1 PEAKS 32 only PEAKS 32 only 26 2 PEAKS 11 only 25 1 PEAKS 22 only 34 7 PEAKS 22 only 52 3 Peptide Number Venn Diagram Protein Number Venn Diagram PEAKS 11 PEAKS 29 PEAKS 11 PEAKS 22 PEAKS 32 PEAKS 32 67 14 Advanced Configuration and Environment Preferences 14 1 PEAKS Environment Preferences This section will describe the setting up the configuration of environmental preferences including general instrument search engine and ion editor configurations To begin click the Preferences toolbar icon Or Select Preferences from the Window menu The following window will open A Preferences puse General
102. h orbisample window differs A a E a a a E from the other Data Refine Segment Match Non gapped Homology Match Homology Match window as it asks De novo you to select a does not give you any filter options General Options PEAKS Search Mass Tolerance Da 0 1 Report Top if SPIDER Search Leucine equals Isoleucine Lysine equals Glutamine PTM Finder PTM Options Quantification Name Mono mass Residue site CJ Fixed Modification In this tuse ve Acetylation N 42 0106 ADCEQGILMP lodoacetic acid derivative Acetylation K 42 0106 K ipd bs caen Amidation 0 984 x c have de novo result Applied Biosys 442 225 C Variable Modification Applied Biosys 450 2752 C amp Deamidation for the data file Applied Biosys 227 127 C Oxidation M Applied Biosys 236 1572 C M C Show unimod Max variable PTM per peptide E By S electin g a Database Options database SPIDER Select Database swiss lal species will Se arch the de novo sequences already generated for that data file that have used the database that you selected 3 The following section will describe the different options that you have when setting up the parameters for your SPIDER search Query Options Choose a Query Type They are in order of increasingly rigorous analysis Segment Match this 1s not a true mutation
103. he Filter Pane Default Parameters orbisample Each filter can be applied several times over So it can get a little complex To illustrate here are a few examples 1 Goal Show proteins that have two high scoring hits Add the Protein Filter called Query and in the Edit Filter section choose greater than and type I in the box without the quotes This will remove any one hit wonders Add the Peptide Filter called Score and in the Edit Filter section choose greater than and type 50 in the box without the quotes 2 Goal Find a protein that contains the word human or rat in the database entry s description but not Keratin or Trypsin Add the Protein Filters called Desc In the Edit filter section you are required to type in a regular expression regex This allows you to use wildcards Wildcard Meaning Example p Anything of any noth human Will find anything that contains the word human with anything before and anything after Or use brackets J human rat RAT Will find anything that contains the word human or the word rat or the word RAT with anything before and anything atter P Not use brackets Keratin Trypsin human rat will find anything containing human or rat but not Keratin or Trypsin Any of these characters Hh uman Rr at will find anything
104. he common instrument types example FT TRAP or specify the instrument vendor and chose the vendor specific instrument type example LCQ Ion Trap Hold down the Ctrl key to select additional instruments Notice that when you select the instrument type the default parameters will be displayed in the ri ght han d Steps Instrument Details pane Select Project Properties Which instrumentist was were used to produce these data the Add a new Sample Properties instrument Bee cee ier Hrhermiscseientific wl Instrument Details LEG Fleet Ion Trap button if your Ira LEQ Fleet Ion Trap a Ton Source ESI nana spray MS Scan 3D Ion Trap instrument 1s LILC Fleet Ion Trap etd Frag type CID CAD IRMPD y and b ions not on the list oO Ecole Ten Tiaria F Mn 3D Ion Trap Lastly select Default Parent mass tol 0 5 da E LTQ FT Ultra Hybrid FT FT Default Fra gment tol 0 5 da osa S e n LTQ FT Ultra Hybrid FT FT e MS centroid or MSMS centroid data has been LTG FT Ultra Hybrid FT FT el centroided Click the F LTG FT Ultra Hybrid FT Trap E n M Next button When only one instrument 1s PAM uw starts loading data ICI LTO FT Ultra Hybrid FT FT pi 7 If more than two instruments are selected Steps Instruments and Files Project Properties This list contains all the files you selected For this proj
105. himi Project to access MassLynx libraries and convert the data PEAKS provides a version of wolf exe compatible with MassLynx 4 1 If you need a different version of wolf exe please visit www bioinfor com products peaks support watersmicromass php Additionally you must make sure that the following MassLynx libraries are installed on the same computer as PEAKS and wolf exe 33 DACServer dll Genutl dll MetaGD32 dll raw dll security Access dll securitySettings dll securitySignature dll ABI 4700 or 4800 Data BSI has created a converter to extract the data from an ABI Oracle database If you require this separate free tool contact your sales representative Once installed you can start up the ABI 4700 Data Extractor from the Start menu System Requirements This extractor can be installed on the same machine as ABI 4700 Explorer and the Oracle database we will call this machine the 4700 SERVER in the following instructions or another machine that has direct network access no firewall or proxy required to the 4700 SERVER Windows 2000 or Windows XP is recommended for use with this tool Configuration Before using the ABI 4700 Data Extractor it must be configured To do so choose Settings from the File menu Configuration requires the following 4700 SERVER Name or IP Address input localhost if the Extractor is running on the 4700 SERVER this 1s the default value otherwise enter the IP address of the 4700 S
106. ic composition the other cell population is fed amino acids labeled with heavier isotopes The heavy amino acids are incorporated into newly synthesized proteins eventually completely replacing the cells proteins such that labeling efficiency is near 100 The cell populations are then mixed together and digested for MS analysis to determine differential protein abundances I Creating a project Click on the Create new project icon m or select New project from the File menu The following window will appear A New Project Steps 1 Project Properties Ze A New Pro ject Steps 1 Project Properties 2 Sample Properties 3 Project Properties Project Name SILAC Sample Project Location C Peaks Studio 5 0 derbyServer serverDB Project Folder Jeaks Studio 5 0 derbyServer serverDB SILAC Sample Notes Description Type and organization of project Basic Project Sample Properties Give this sample a name Select Files Name SILAC 1 Size Date Modified Give your project a name such as SILAC Sample Then click Next The following window will appear Give your sample a name such as SILAC 1 Click the Add a file for this sample button and select the file C PEAKS 5 Data SILACSample mzxml Click OK to add this to the list of selected files For cases where you want to add another sample to the project select Add another sample and repea
107. ick on the Chart View tab The following window will appear gdssenqalgpaqpnqddkadvqtdagclsepvasalipvkdhkllekedseaadsksLhvl Protein Clusters Info Protein Score Distribution Chart 100 Multi detection clusters 18 90 Two detection clusters 15 80 Single detection clusters 70 32 Peptide score threshold 202 Calculate 60 50 4 Chart View Protein View Peptide View Denove view amp o o eo e a o In 40 4 30 20 10 4 o Protein Cluster Index False Positive Rate Chart amp amp False Positive Rate Peptide Score Peptide Index Decoy Search Result Peptide Score Distribution Chart Score Threhold 95 Forward Peptide Ma Reverse Peptide Ma False Positive Rate 50 60 Threshold 61 This feature will be described using the data that was chosen for the walkthrough as it is simple data The Protein Score Distribution Chart shows the distribution of the protein scores by percentage The default peptide score threshold is 20 In the above example this threshold results in 32 proteins with a single detection cluster 15 proteins with two detection clusters and 18 proteins with multi detection clusters Modifying the peptide score threshold using the up down arrows and clicking on the Calculate button wi
108. ied Type Sample iTRAQ mzXML 4 352KB 01 22 2009 12 12 44 PM mzxml The following section will tell PEAKS which type of mass spectrometer was used to generate the data This sample was derived i ple Properties fr oma Quad TOF Click the box Instrument Details General Instrument Details Quad TOF beside Quad TOF to select this E FJuneer Trap pa ton Source ESI nano spray instrument Flousdrt ibi isis Quad FT ecd MSn Scan Time of Flight TOF A New Project Steps Instrument Details Project Properties Instruments s used to acquire the data Default Parent Mass Tolerance 0 1 Da C Quad Quad Default Fragment Mass Tolerance 0 1 Da MS Data Centroided yes FF TOF TOF MS MS Data Centroided yes Upon clicking Next a sample ee 1 project will be created Your aiii a Main Processing Screen should look something like this Fragmentation Type CID CAD IRMPD y and b ions no no 110 2 Running data refinement In the Project View Frame select YTRAQSample mzxml Click the data refinement tool b or select Data Refine from the Tools menu Enter the following parameters shown below and click OK A Data Refine Tools Y Data Refine De novo PEAKS Search SPIDER Search PTM Finder Quantification Charge Options Filter Options 111 c J cw J 3 Running PEAKS Search Click the PEAKS Search t
109. ill in your details in the Basic Options panel Instrument Details In the manufacturer drop Instrument Name down list select gt specific p e ME Agilent Technologies v vendor Or General Ion Source cR 3 MS Precursor Scan 3D Ion Trap v Fragmentation Type CID CAD IRMPD y and b ions v MSn Product Scan EE Ion Trap v Advanced Options Precursor mass search type Monoiostopic O Average Parent mass error tolerance da v Fragment mass error tolerance da v 103 Ion Source Use the drop down list to select what ion source that was used MALDI SELDI or ESI nano spray This will help the PEAKS Data Refine tool to decide the charge of the ions MS Precursor Scan Use the drop down list to select what type of MS scan was performed This selection will tell the PEAKS Data Refine tool if the survey scan is of sufficient resolution to determine the charge and the monoisotopic peak from the examination of the survey scan Fragmentation type Use the drop down list to select the method of fragmentation that was used This selection will tell PEAKS what type of 1on series to expect for PEAKS auto de novo sequencing and PEAKS protein ID database search Select CID ECD if alternating fragmentation is used to allow the algorithm to determine the type of fragmentation from each scan header MS Product Scan Use the drop down list to select what type of MS scan was performed This selec
110. inear Ian Trap FT trap tecd cid Default Parent Mass Tolerance 0 01 Da Default Fragment Mass Tolerance 0 5 da MS Data Centroided C yes MS MS Data Centroided O yes FTMS ecd cid FTMS etd Add a new instrument We will leave the instrument vendor as General and select the instrument to be FT trap Click Next You should now see the file in the Project View panel The Project View panel which is shown PA Project View in the upper left hand corner displays the c M CU Peaks Studio 5 0 derby Server serverDBIMew Project 1 qe Tnm organization of a particular project Use Eh Jk Sample 1 e 9 L OrbiSample mzXML the and boxes to expand and collapse the project in order to access the data file in the Project View Make sure that you select this data file when choosing data to be analyzed 14 PEAKS reads and tracks information about the experiment and data for use in the analysis and for future reference Once the data file has loaded click on the Properties tab in the bottom left hand corner Tasks Running Info Properties Selection Details Orbi amp ample mzxML Total MSI Spectra 4 Total MS MS Spectra 6 Ton Source ESI nana spr ay Fragmentation Mode CID CAD IRMPD Cy and b ions MS Scan Made FT ICROrbitrap MaMS Scan Made Linear Ion Trap MS Scan Centroid False 5 5 Scan Cenkroid False You should see the following in the Main Processing
111. ing over the spectrum will display a tooltip in the new window that will display the m z ratio and the height intensity as a percentage of 100 of that particular peak Both the m z ratio and the height of the peak can also found under the spectrum view on the right hand side To zoom either on the X or Y axes select the Zoom X or Zoom Y buttons respectively and then use the wheel on your mouse to move around the graph Selecting the 1 1 button will restore settings to view the entire spectrum on the screen You can use the profile and peak ul buttons to switch the spectrum view from profile mode to peak mode and vice versa The scrollbar on the left acts to increase and decrease the intensity of the peaks where the scrollbar on the right acts to zoom in to display the monoisotopic peaks 49 Spectrum Alignment Frame Info Survey Alignment Error Map 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 Clicking on the Alignment tab will display the Spectrum Alignment Frame This frame always shows the whole spectrum and is used as a tool to help us navigate the spectrum view frame A blue bar along the horizontal axis of the alignment view indicates the range of the spectrum view in the Spectrum View Frame This frame will show you how the proposed ions align with the spectrum By default the Spectrum Alignment Frame displays b ions and y ions The b ions are shown
112. is allows you to open a new data file created by the mass spectrometer in its native format or a PEAKS data file in ANZ format that also contains peptide analysis data Other accepted file formats include PKL DTA MGF or ANZ al Close File Selecting this icon will remove the presently selected data file from its project 123 New Project Clicking the New Project button will allow users to create a brand new project offering organization to any study m Open Recent Project The quickest method to recalling an existing project Users are instantly directed to a selection of recently created modified or viewed PEAKS projects Close Project When a project is complete or a user temporarily no longer is working on a particular project users can click this icon to remove it from their Project View Save As The project method of PEAKS allows for all results to be automatically constantly saved However if a user wishes to save a particular application under a new heading this is the most convenient and appropriate way to do so Just click the icon and enter the new title in the field provided The file will be saved in the ANZ format Press this after selecting a data file in the Peptide Data Frame Print Whether a user desires to print their ms spectrum views to complement a publication or print matrices the print feature offers a straight forward connection to any printer configured to the user s computer g Export Easily expo
113. ist 4 After setting up parameters we can save them for future use Click the Save Parameters button and choose a name for future reference when prompted Any parameters that you save will be available in the drop down list at the top of the window To see what s inside just select one and the parameters boxes will be populated 5 Press the OK button If you have already performed de novo sequencing the database search will commence automatically If you have not previously performed de novo sequencing the auto de novo process will appear first in the task queue Once de novo sequencing is finished the database search will begin 54 7 2 Protein Identification Results Peptide View Once PEAKS is finished searching the database the Peptide View window will open by default F ID Sequence PEAKS Scor RSD Miz Z Mr Calc DeltafMa Error ppm File RT Scan Quality InChoru Reconst gt EEE o J Peptides Spectrum 1 DNPOTHYYAM 99 0 0 0 802 91 2 1603 7993 0 0061 3 8056 OrbiSample m 0 072 0 788 99 0 Spectrum 2 TSDANIN 2 W 99 0 0 0 695 84 2 1389 6523 0 0132 2 4869 OrbiSample m 0 314 0 785 99 0 F Spectrum 3 ANELLINVK 97 37 0 0 507 3 2 1012 5916 0 0062 6 0879 OrbiSample m T 0 365 0 781 97 37 r Spectrum 3 ANETLINVK 97 37
114. ition to reading this manual we recommend that you take a look at our training videos that explain the main features of PEAKS in detail http bioinfor com products peaks support tutorials php Please send technical questions to support bioinfor com We also encourage our users to provide us with any suggestions or comments as we are always improving PEAKS to meet the needs of the scientific community http www bioinformaticssolutions com corporate contactform php 2 Getting Started with PEAKS 5 This section of the manual will guide you through the installation and configuration of PEAKS 5 2 1 Package Contents The PEAKS 5 package contains This manual hardcopy and or electronic version PEAKS 5 software 2 2 System Requirements PEAKS 5 will run on most platforms with the following requirements Processor Equivalent or superior processing power to a Pentium III at 800 MHz Memory 1 GB minimum 1 5GB is recommended for PEAKS Studio PEAKS Client SOOMB is recommended for PEAKS Viewer Operating System PEAKS runs on Windows Vista XP and Linux Adjusting the Amount of Memory that PEAKS Uses The PEAKS Studio directory e g C PEAKS Studio 5 0 contains a file called StartPEAKSStudio lax which contains a line which looks like lax nl java option java heap size max 1200m The 1200m tells the Java VM which runs PEAKS to run with 1200MB of memory With some trial and error the Java VM will fail to run
115. ixed modification selecting a post translational modification as a fixed modification tells PEAKS that this modification 1s applied to all occurrences of the residue s on which the PTM can occur Enzyme Biomolecules that catalyze chemical reactions including the digestion of proteins ESI Electrospray Ionization A method for ionizing a sample into the mass spectrometer m z mass to charge ratio MALDI Matrix Assisted Laser Desorption Ionization A method for ionizing a sample into the mass spectrometer This has a characteristic effect of producing singly charged ions Mass accuracy this refers to the accuracy of data obtained from a given mass spectrometer On a spectrum this is reflected by how close the peaks are to the actual masses of the ions they represent PTM Post Translational Modification A newly translated protein may differ from its final form as a result of processing by various enzymes in the cellular environment This change is referred to as a post translational modification Since PTMs change the mass of residues it must be accounted for when sequencing peptides by mass spectrometry Built in PTM PEAKS comes equipped with a library of possible post translational modifications These can be incorporated into a de novo analysis at the click of a button Customized PTM If the post translational modification you are looking for is not in the PEAKS PTM set you may create our own entry or modify an existing one This
116. le cleavage C ves ng predefined methods 1 9 Method Select device amp parent Sm Fi Quad TOF 100 ppm Quad TOF 0 5 Da Ion Trap 4 Da 26 Finally check the OMSSA box and select the name Enter the following settings InChorus Search OMS5A PEAKS Protein ID e NCBI O M SSA C x Tandem IE Search Search Status Hel Mascot Enzyme Trypsin Maximum missed cleavages 3 Species to search all species Sequence library aa kw i aua Aeropyrum pernix Sample DB Methanococcus jannaschii Taxonomy supported Other Archaea Bacteria Actinobacteria Sequest C Import Result Hitlist max length 10 Fixed mods ctrl key for multiple selection carboxyamidomethylation of K E value cutoff 1 Variable mods ctrl key for multiple selection citrullination of R deamidation of N and Q dehydro of 5 and T citrullination of R di O18 on peptide n term deamidation of N and O di iodination of Y dehydro of 5 and T di methylation of K idi O18 on peptide n term loxidation of M carbo nurenin of W carboxymethyl Maximum variable mod combinations searched per peptide 64 Precursor mass tolerance Da 0 1 Product mass tolerance Da 0 8 Precursor mass search type monoisotopic Product mass search type monoisotopic Lower bound of precursor charge 1 Upper bound of precursor charge 3 x Minim
117. lerance Precursor Mass Search Type Preprocess this data on the fly dialogue Fragment Mass Error Tolerance Monoisotopic C Average Max Missed Cleavages 3 window will Enzyme Options appear 2 Mew Enzyme Digest Rule Advanced PTM options Er Fixed Modification 4 amp Iodoacetic acid derivative Residue site ion tM 42 010567 ADCEQGILMPSTV amp N Acetylation K amp 2 010567 IK o Amidation fosso eG Applied Biosystems ari 442 225 Mono mass Variable Modification 4 2 12698 2 pplied Biosystems cle pplied Biosystems cle 236 15718 3 To change pnlied Biosystems iTR pr otein Database Options identification 3 Select database fede all species elect Database Swiss Pro search Paste fasta sequences Edit Database ew Database parameters cuc DOW is the Set Taxa time Advanced Options PEAKS uses a hybrid search technique that requires some sequence tags to help in the search D ill I have already run de novo don t run it again Run de novo using different parameters than the above Run de novo using the same parameters as above default validation decoy search Mass Options Parent mass error tolerance Determine how much random and systematic experimental error on the parent precursor 10n PEAKS will allow for in its analysis Type a tolerance in the textbox and choose units from the dropdown list Using PPM allows for larger errors at l
118. ll result in changes to the amount of clusters that are found for each protein The Peptide Score Distribution Chart displays the scores of the individual peptides as a percentage The False Positive Rate Chart is derived from running a decoy database search which can be selected from the Advanced Options panel when you are setting up your Protein ID parameters Advanced Options PEAKS uses a hybrid search technique that requires some sequence tags ko help in the search have already run de novo don t run it again Run de nove using different parameters than the above Run de nove using the same parameters as above default The example shown above indicates that below a score threshold of 30 there is a false positive rate of approximately 1 More specific details about the false positive rate can be seen in the Decoy Search Result table For example a score threshold of 20 resulted in 88 matches using a forward database search and 8 matches using a reverse database search The false positive rate was therefore 8 33 62 8 SPIDER Search After having obtained de novo sequences for peptides that are not in the database it s a good idea to look for a homologous peptide in the database This will help you to learn more about the proteins in your sample To search with SPIDER you must first have some good de novo sequences 8 1 Setting up SPIDER Parameters 1 Select a data file or a Protein ID resu
119. lt from the Project View frame 2 Click the SPIDER icon on the toolbar Or Choose SPIDER Search from the Tools menu When a Protein ID results file 1s selected the SPIDER Search Options window will appear as seen below A SPIDER Search Query Options Data Refine Segment Match Non gapped Homology Match Homology Match De novo General Options PEAKS Search Mass Tolerance Da Report Top SPIDER Search Leucine equals Isoleucine Lysine equals Glutamine PTM Finder PTM Options Quantification Name Mono mass Residue site Add Fixed gt Fixed Modification Acetylation N 42 0106 ADCEQGILMP Acetylation K 42 0106 K E Amidation 0 984 xJ c Applied Biosys 442 225 C MM Applied Biosys 450 2752 C Applied Biosys 227 127 C Applied Biosys 236 1572 C v C Show unimod New PTM Max variable PTM per peptide Filter Options Use the spectra which satisfy the Following conditions For use in the SPIDER search De novo amino acid score ALC greater than i recommended D 5 Protein ID peptide score less than recommended 0 65 De novo Options Advanced Spider search requires sequence tags generated by de novo sequencing For the search Use existing de novo results If you have selected a data file the following window will appear SPIDER Search Note that this SPIDER Searc
120. m Settings Clicking on Search Engine and then X Tandem Settings in the menu on the left hand will open the following window x amp Preferences General XTandem Settings Instrument Search Engine Launch Server Local Search Ha P nE XTandem Server Settings Se XTandem Settings i Sequest Settings Ion Editor B Basic Ion Editor N Advanced Ion Editor XTandem Local Settings First you must select whether you would like PEAKS to access a server or local version of X Tandem If you select the server version you must enter the Host name or IP address as well as the port To make sure that you entered everything correctly and that the server 1s working click the Test Connection button If you select a local version of X Tandem you must click the Browse button to tell PEAKS where to find the local settings If PEAKS provides the local copy uses the location of PEAKS as the default path Click the Apply button to save any changes you have made 94 OMSSA Settings Clicking on Search Engine and then OMSSA Settings in the menu on the left hand will open the following window amp Preferences Omssa Settings Default Omssa Path uu Mascot Settings OOS Tandem Settings E Omssa Settings Search Engine m Sequest Settings Ion Editor To use OMSSA you must click the Browse button to tell PEAKS where to find the default path If PEAKS p
121. mat associated with Mascot software ANZ the zip compressed XML based file format associated with PEAKS 4 5 XML format files using the mzXML schema XML format files using the mzData schema RAW files from Thermo Electron instruments WIFF files from ABI Sciex QSTAR and QTRAP instruments RAW files from Waters QTOF instruments BAF YEP and folders of FID files from Bruker instruments D files from Agilent QTOF instruments DAT files created by BSI s ABI converter software PEAKS 5 project 4 2 Data Conversion It is best to import RAW data directly so that PEAKS can access the complete unprocessed experimental data including the MS survey scan and retention time information This will ensure that the PEAKS analysis does not suffer from poor preprocessing In order to load RAW data from different vendors PEAKS may require third party software to be installed Please consult the following instructions for third party software requirements Thermo Data RAW data from Thermo Electron mass spectrometers can be loaded provided that the XCalibur software is installed on the same computer as PEAKS 5 Agilent Data PEAKS 5 can load native data from Agilent QTOF provided that the MassHunter software is installed on the same computer Bruker Data PEAKS 5 can load data from Bruker mass spectrometers provided that the CompassXport software 1s installed on the same computer If loading fid files which are stored in a network of fol
122. med in Frag Mode Mode that the fragmentation step was performed in The columns themselves can be customized Right click anywhere in the report and choose Toggle Column from the pop up menu The sub menu that appears shows a checkmark in each of the columns that are currently showing Click any one of them to show or hide a column These settings will apply to all your reports Peptide Alignment Click on the Peptide Align window This will look very similar to the de novo results window You will see the Ion Table which shows the proposed ions with their corresponding masses To the right of the Ion Table is the Error Plot which displays the confidence that is assigned to each ion The most confident results lie on the centerline Clicking a cell or column in the Ion Table highlights the corresponding points on the error plot and corresponding peaks on the spectrum Underneath the Ion Table is the Spectrum View Frame which displays a graphical representation of the spectrum The peptide that corresponds to the spectrum in the Spectrum View Frame is displayed in the Input Sequence box Note that this 1s a drop down menu so that you can select other peptides that have the same ID if applicable Scrolling over the spectrum will display a tooltip that will display the m z ratio and the height intensity as a percentage of 100 of that particular peak Both the m z ratio and the height of the peak can also
123. n ID result file and click the PEAKS Quantification toolbar icon Q Or selecting Quantification from the Tools menu The quantification parameters window will open Enter the parameters as shown below and click OK amp Quantification Quantification Basic Options Data Refine Mass Error Tolerance 0 1 Da v Upper Bound of Precursor Charge 43 De novo Retention Time Range 1 0 min w PEAKS Search Label Options SPIDER Search C Labelling occurs at the M5 M5 level eg iTRAQ Labelling occurs at the MS level eg ICAT PTM Finder TEN Lm Quantification Sample Added Mass Residues Labelling Efficiency t Heavy 5 0 K 1 0 Light 0 0K Ali Add Label Delete Label Once completed the protein quantification result will be displayed in the same PEAKS Protein ID result window that you selected earlier The results are listed as a Ratio Heavy Light and Standard Deviation Heavy Light They are highlighted in the red box below For example the highest ranked protein Human Filamin A has a ratio of 1 2 and a standard deviation of 0 1 amp Accession ID Mass Display PEAKS Score 96 Coverage Ratio Heavy Light SD Heavy Light Query matched Marked Description 5 P21333 FLNA_HU 61 280736 56 98 95 2k 1 18 0 1 6 I Filamin A Alpha filamin p P04264 K2C1_HU 62 66017 65 BN 34 7 T o o 3 E Keratin type II cytoske 2 Q9Y283 INV
124. na DOE cen ueuns accuses TREND IE danse IE ER e dae 65 Fiter ODOlS oai ete ie ir cet ae tae rede Ol cedat iiid 65 De novo Secr 66 PI PLPETRSURIDICI ETC ELT LE 66 9 2 SPIDER Results VIOW ieeeccti rex EE ate eee a x Deka Eee adu es e PROP esie sx Tue TE 66 9 WP Wine 68 9 1 Setting up PTM Finder Parameters ccccccccssssssssssssscccccscssssscsssccccsssssssssscees 68 92 P TM Finder Results VIeW 5 dede DE d o uev deve p ee rnc ouod 69 10 mChor s Meta Search aeree PRSE QUEE ER RESO EEETEeE ERI IEEE V Haa Te ER Roadie 70 10 1 Setting up inChorus Parameters e eee eee eee eee ee eee eee esee eese sss asne 70 Importing Existing Results cecinere INIHI EE EI Ene FIER PORE IUD EDIEEEEEE 71 10 2 inChorus Results View ccsesesececccecececececececececececeoeseocccocococococococococococococococococososo 72 De novo Peptide and Protein Views ssssscececcossssscceecccossssecceocoossssceeeoosossssseee 72 Chart VIGW o ooteatine iin avete b iot tn 72 li Filtering Your Results REPE SERRE EE EO ERR Peia Ree re Ee Ee ERE dS 73 TEE Setting Filter Parameters anus ERE e eR EN EE EVES EUER ET Se iN NEI 73 Possible Filters Selected Filters Edit Filter 74 Filter ODLUODS iie edle a EE REEF khoe ecce vivi inei e abba cca t idis 75 Parameter OpDUODS e siete loire eben ae
125. ng PEAKS protein ID it is helpful to enter a peptide score threshold so that SPIDER will only be performed on peptides below the threshold The recommended threshold is 0 65 65 De novo Options Because SPIDER requires a de novo sequence to find homologous proteins in the database de novo sequencing will need to be performed first If you have already done de novo sequencing select the I have already run de novo button Database Options Note that these options are only visible if you choose to run a SPIDER search on a data file rather than a PEAKS results file Database to search Select from this dropdown list one of the FASTA databases configured in PEAKS To edit an already existing database click on the Edit Database button If the desired database is not in this list click the New Database button Note that you can also set up a new database in the Database Configuration window The configuration window is the only place that that you can delete databases that you have created For more information on setting up new databases see page 99 Taxonomy selection This list displays the taxa you have chosen for your search If the database selected has taxon information available you can click on the Set taxa button Otherwise the whole database will be searched The selections correspond to established hierarchy 1 e selecting Mammalia will search all of horse cow rat mouse human etc 4 Afte
126. ntitled to a perpetual license select the Request License file and click Next In the field that appears enter the registration key that came with the product whether it is a key for the full version You must also enter your name the name of your organization An automated servant from BSI will generate the license file license lcs and email it to the provided email account from the License Wizard Save the license file to a local directory and then continue with the License Wizard to import the license file into the PEAKS folder A dialogue box will then read Registration Successful A License Wizard License Wizard Please select one of the Following tasks Import License File I have the license File Backup License 5 Update license Request a 30 days evaluation license If you are not connected to the internet onscreen instructions will offer assistance for offline registration Re registering PEAKS may be necessary if your license has expired or if you wish to update the license You will need to obtain a new registration key from BSI Once you have obtained this new key select About PEAKS Studio from the Help menu The About BSI PEAKS Studio 10 dialogue box will appear Press the License Wizard button to continue Follow the on screen instructions About BS PEAKS Studio PEARS Studia 5 0 build 20081107 Copyright 2000 2009 Bioinformatics Solutions Inc All rights reserved
127. odification box If you drag over an incorrect PTM simply drag it back to the PTM Options list Max variable PTM per peptide To reduce uncertainty limit PEAKS de novo sequencing vocabulary by restricting the number of variable PTM found on a peptide Specify a number by typing it into the box To lift such restrictions type a very large number longer than the length of the peptide General Options Report up to peptides Set how many peptide sequences PEAKS will report from its de novo sequencing analysis 45 Preprocess your data on the fly before auto de novo PEAKS has its own built in preprocessor for removing noise centroiding and deconvolution Check this box to turn preprocessing on BSI highly recommends using PEAKS to preprocess all data as opposed to using instrument vendor software if the data is to be used by PEAKS PEAKS preprocessor should not be used on data that has already been pre processed as this will have adverse effects on the results unless it is 10n trap data Note If you have already pre processed your data in the data refinement step you do not need to do this again 4 After setting parameters you can save them for future use Click the Save Parameters button at the top of the window and choose a name for future reference when prompted Any parameters that are saved will be available in the drop down list at the top of the window To see what s inside select one and the parame
128. og by default Select the Browse button to change this location RMI Connections Clicking on RMI connections on the menu on the left hand will open the following window amp Preferences F General RMI Connections RMI Connections H Server Host S ial Derby Database ti PErtOIan C Server Port 3003 H Instrument MEM E 4 Search Engine Client Park 1000 i Ion Editor a Worker Port 5000 The default port numbers for the Server Client and Worker will appear The port numbers can be changed if conflicts arise Contact technical support at BSI for more information Click the Apply button to save any changes that you made 89 Derby Database Clicking on Derby Database on the menu on the left hand will open the following window amp Preferences Eg E General Derby Settings s RMI Connections SOSE Derby Host ocathost 3 Performance zer E instrument Memory used to start Derby Server 1024 i Search Engine Derby Jar Location Ion Editor C org apache derby core_10 2 2 _ User Define Browse Derby Host The name of the Derby Host as well as the Port number will come up by default and can be changed if needed Memory used to start Derby Server The amount of Memory used to start Derby Server will also come up by default but can be changed if more memory is available D
129. old is 0 65 9 2 PTM Finder Results View The results from a PTM finder search are identical to those seen in a PEAKS Protein ID search Please see page 55 for more information on the PEAKS Protein ID search results 69 10 inChorus Meta Search inChorus Protein Identification will call upon several search engines for protein identification and will then compare and summarize the results from the different search engines in one single report PEAKS protein ID X Tandem OMSSA Mascot and Sequest Please note that you will need to have your own copy of Mascot and Sequest in order to make use of those search engines during and inChorus search In order to set up your search engine preferences see page 93 10 1 Setting up inChorus Parameters 1 Select the orbisample mzxml file 2 Click the inChorus Search icon on the toolbar Q Or Select inChorus Search from the Tools menu The inChorus Options window will appear InChorus Search Tools PEAKS Protein ID C X Tandem omssa C Mascot C Sequest Import Result 3 First select each of the protein identification tools that you would like to use by putting a checkmark in their respective checkboxes Search parameters for each program can be set by selecting the name of the search engine You will need to use the Ctrl button to be able to check the boxes for multiple search engines The option screens for each of the search engines available to inChorus
130. on 42 047 CKRHDENQ Myristoylation 210 1984 KC G J N N acyl diglyceri 788 7258 C N isopropylcar 99 0684 C N Succinimidyl 127 0633 K X J N Oxidation M 15 9949 M Oxidation HW 15 9949 Hw Palmitoylation 238 2297 IcsTk y p T L C Show unimod New PTM Max variable PTM per peptide 3 Filter Options Filter the spectra which satisfy the Following conditions For use in the PTM search De novo amino acid score greater than 0 5 recommended 0 5 Protein ID peptide score less than 0 65 recommended 0 65 lt 68 The parameters are the same as you used when performing protein ID page 51 with the exception of the filter options found at the bottom of the window As PTM Finder searches tend to be computationally intensive PEAKS will only look at de novo sequencing results that are above the amino acid score threshold and below the peptide score threshold that you input De novo score A A threshold The PTM finder requires a good sequence tag from de novo to be able to find good quality homologous proteins Enter a value for the de novo score threshold The recommended threshold is 0 5 Peptide score threshold Because there is no need to run the PTM finder on peptides that were already found to have a good match during PEAKS protein ID it is helpful to enter a peptide score threshold so that SPIDER will only be performed on peptides below the threshold The recommended thresh
131. on PEAKS Protein ID A Mass Options General Options Parent Mass Error Tolerance Precursor Mass Search Type Preprocess this data on the fly Fragment Mass Error Tolerance Da o Monoisotopic Average Max Missed Cleayages ET Enzyme Options PTM Options Mona mass Residue site Add Fixed gt B Fixed Modification E 111 0416 K A Todoacetic acid derivative ICPL light 105 0215 K X amp N Todoacetic acid 56 0055 c Lipo 188 033 K Methyl ester 14 0156 BE 2 Add variable gt D Variable Modification Methylation 14 0156 CKRHDENG O18 label 2 0042 STY XI C Show unimad New PTM Max variable PTM per pepzide Database Options Select database Select Database a Sample DB w SetTaxa e 4 7 Paste fasta sequences advanced Options PEAKS uses a hybrid search oe that ee same sequence tags to help in the search C Run de nova using different parameters than the above C Run de novo using the same parameters as above default Validation decoy search 19 Parameters can be saved and used for future reference by clicking on the Save Parameter button For more information on setting up protein identification parameters see page 51 Click OK to commence analysis After PEAKS Protein ID is completed the click on the De novo view tab Recall that PEAKS found de novo sequencing results for all six spectra however only four spectra ID 1
132. on types and their charges then add them to the ion table calumn list Ton Types Ton Table Columns Ton Editor To select an ion type to be viewed in the ion table click on the ion type in the Ion Type list found on the left hand side of the window You now need to select the charge for that ion type from the drop down menu Once you have done this click on the gt Add with charge button and the 1on type will now appear in the Ion Table Columns list on the right hand side of the window To remove an ion type from the Ion Table Column list select the ion type and click on the Remove from list lt button The ion type will now appear in the Ion types list Click the Apply button to save any changes you have made 96 14 2 PEAKS Configuration This step includes configuration of enzymes PTMSs databases instruments and parameters To begin click the Configuration toolbar icon 5 Or Select Configuration from the Windows menu Enzyme Configuration PEAKS can use almost any enzyme or combination of enzymes in your analysis You can select from any of the built in enzymes or define your own From the Configuration window select Enzyme from the left hand to change your enzyme configuration Built in enzymes All of the built in enzymes within PEAKS are listed in the Enzyme list Clicking on one of these built in enzymes will display the information listed about that enzyme in th
133. onal confidence Finally at the bottom of the window you will see the sequence of the selected protein and in blue you will see where the selected peptide matches the protein The darker the blue the more confident the match is The matched peptides will be shown in red if you have performed a SPIDER search which is discussed in the next section Protein View Click on the Protein View tab The following window will appear E Accession ID Mass Display PEAKS Score Coverage Query matched Marked Description o DB Search F HE P00722 6GAL 1 1164828 corru 3 r Beta galactosida O Q29443 TRFt 2 777532 M H gg 99 15 15 48 15 F Serotransferrin p m PO2769 ALBL 3 69293 55 EEI 99 1 14 99 14 F Serum albumin pr v a A7ZUEO GLPK 4 56230 816 I il I 98 81 7 97 E Glycerol kinase gg P49064 ALBL 14 68659 56 i 98 52 7 57 5 F Serum albumin pr ded amp Q28522 4LBL 27 67831 09 Nn 91 41 5 67 3 Serum albumin pr i m POD69s LYsc 29 16238 6388 Em 83 72 17 69 2 F Lysozyme C prec c i m Q4UZR8IGLPI 31 552295 83 72 4 41 2 C Glycerol kinase 2 i m P00330 ADH 49 36823 1527 T 83 69 5 17 2 Alcohol dehydrag aid POF724 4LBL 52 68692 59 I 70 87 4 11 3 Fj Serum albumin pr He o Q9Y7O7 ACT 60 41663 453 a 60 7 7 47 1 F Actin 2 Suillus b lt Q1IP56 OPN 63 54489 82 E 60 7 4 8 2 Fl Melanopsin like m POO7O6 LYSC 64 14507 404 EJ 50 7 11 63 1 Lysozyme C 3
134. oolbar icon ca or select PEAKS Search from the Tools menu The Protein Identification Parameters dialogue window will appear Input the parameters as following A PEAKS Search fc aft Tools Database Search ITRAQ w Mass Options General Options Data Refine Parent Mass Error Tolerance 0 3 Da w Precursor Mass Search Type Prepracess this data on the fly Fragment Mass Error Tolerance o Da Monoisotopic O Average Max Missed Cleavages 3 Y PEAKS Search is pirum Ie z cone Tes J SPIDER Search B NO l Digest Rule RE P WIESE Ch Ane PTM Finder Find peptides that ssrishythe above rule at both end Quantification PTM Options Wn Residue ste 3 Feed Modesto TE N 42 0106 ream m e Applied Biosystems iTRAQ TM 4plex N Acetylation K 42 0106 p Rem Applied Biosystems iTRAQ TM 4plex K Amidation 0 984 i Carbamidomethylation Applied Biosyst 2 225 E B Applied Biosyst 450 2752 Add Variable gt e Variable Modification Applied Biosyst 227 127 c Oxidation M une aer r pled Bosystens TAQ Ax Applied Biosyst 144 1021 EN Show unimad Max variable PTM per peptide 3 Database Options Select datab e ue Select Database Swissprot w Paste fasta sequences Advanced Options PEAKS uses a hybrid search technique that requires some sequence tags to help in the search Ca L have already run de novo
135. or OMSSA is listed as an E value For more information about different scoring methods refer to the user manual of the 3 party search engines The inChorus search report also looks very similar to the 3nd party search engine results however there are no De novo View and Chart View results available The de novo sequencing results that are found in the De novo View are only those that correspond to results that have been identified by one of the search engines in the inChorus search Chart View As mentioned above Chart View is available in the inChorus report The two charts that appear at the top the Protein Score Distribution Chart and the Peptide Score Distribution Chart are in the same format to those that are seen in the Chart View of a PEAKS Protein ID search page 61 9 In the example below the inChorus Protein Number Pie Chart displays the percentage of identified proteins that were found by PEAKS OMSSA Mascot and X Tandem PEAKS identified 82 of the proteins on its own and 15 of proteins were common between some of the search engines The inChorus Protein Number Venn Diagram gives more specific information than the pie chart about the overlap of the results between different search engines The Venn diagram contains information about 3 search engines and inChorus was run using 4 search engines for this example You can change the search engines that will appear in the Venn diagram using the dro
136. pdown InChorus Protein Number Pie Chart Search Engines lists Select h j h InChorus Protein Number Venn Diagram E iioi only PEAKS XTANDEM engines that you would like to compare and click the Apply button 12 11 Filtering Your Results PEAKS 5 provides you with an exhaustive list of all proteins and peptides that can be found in a sample However since everyone has their own criteria of what information is required in their report and what is an acceptable result PEAKS 5 provides the necessary filtering tools that enable you to filter out the less critical information and leave you with the essentials 11 1 Setting Filter Parameters Click on the time and date stamp associated with the result that you would like the filter Once the report loads click the right button on your mouse and select Perform Filtering The following window should appear Data Refine Tools ID De Novo Filters De Novo Filters r I Peptide Filters Peptide Filters y Result Filtering uw J rotein Filters Protein Filters Filter Options Edit Filter O De novo view shows peptides that could not be explained by proteins from the Peptide View De novo view shows all peptides that are not filtered o Remove de novo peptides with no matching database results Parameter Options Set saved as default Clear default Default Parameters FA Project View i OrbiSample Final Jl Sample 1 EE OrbiSample
137. r setting up parameters we can save them for future use Click the Save Parameters button and choose a name for future reference when prompted Any parameters that you save will be available in the drop down list at the top of the window To see what s inside just select one and the parameters boxes will be populated 5 Press the OK button and the SPIDER search will begin 8 2 SPIDER Results View SPIDER will search the database for homologous peptides and attempt to consolidate these into protein hits as well The result report will look much like the results for PEAKS Protein ID or inChorus searching Clicking on the Peptide View tab will display results that look very much like the results for PEAKS Protein ID See the section on page 55 for more details Click on the Peptide details Peptide Align Peptide Details tab to see the SPIDER matches shown in red Select peptides For display sp Q29443 TRFE BOVIN Denovo DNPQ THYYAVAVVE Letters on a green background and with TITETENIT vertical bars indicate agreement Letters on a Recon DNPOTHYYAVAVVK P HLELLTTTILLLI red background indicate sequencing error HomologDNP QTHY Y AV AV VK Color codes on the de novo sequence letters still indicate positional confidence Letters on 1 MRPAVRALLA CAVLGLCLAD PERTVRWCTI STHEANKCAS FRENVLRILE a blue background indicate uncertainty or mutation signs represent more likely mutations brackets indi
138. rame The peptide candidates are sorted by ID Right next to the proposed sequence the auto de novo Total Local Confidence TLC and Average Local Confidence ALC confidence scores are shown You will also see the m z ratio mass retention time etc listed for each peptide sequence For information including color coding see page 47 Below the Peptide Candidates Frame is the Ion Table Frame Each amino acid is color coded by confidence level see page 47 with the masses for matched a b and c ions listed in blue and for the matched x y and z 10ns listed in red Below the Ion Table Frame is the Spectrum View Frame This frame is useful for seeing the strength of the ms ms peaks that PEAKS 5 has set as ions Here the alignment of the assigned b blue and y red 1ons with the entire spectrum corresponding to the selected peptide can be observed For more information on the Spectrum View Frame see page 49 18 At the bottom of the page is the Error Map which displays the confidence that is assigned to each ion The most confident results lie on the centerline For more information on the Error Map see page 50 3 5 Run Protein Identification 1 Click the PEAKS Search toolbar icon Ei Or Select PEAKS Search from the Tools menu 2 Enter the settings as shown PEAKS Search Tools Data Refine De novo PEAKS Search SPIDER Search PTM Finder Quantificati
139. ratio over MS MS number of peaks after pre processing sum of all peak intensities and length of the longest simple sequence tag that can be generated You can choose a threshold of quality score a value from 0 to 1 for accepting a scan We recommend a quality filter of 0 65 Set to 0 01 to disable quality filtering Preprocessing MS MS Scans This section deals with deconvolution de isotoping centroiding and noise filtering within the MS MS data Preprocessing can save hard disk space or upload time But make sure to have the original data available in case you need to refer to it later To see how your data is changed after data refinement refer to the data properties window 5 3 Data Preprocessing Results 2 rEsTe 221106 CT OTnew HCD 02 RAW To view the result of data pre processing click on 9 wo the MS MS tab on the spectrum view In following aee We example the spectrum m z 473 70502 results d from raw spectra m z 473 71 and m z 473 7 MOa 5B4 732 H 468 75 2 H gt 504 712 43 n 6 De novo Sequencing 6 1 Setting up Auto De novo Sequencing Parameters 1 In the Project View Frame select the data file s or project containing the spectra that you wish to sequence by Auto de novo 2 Click the Automatic de novo toolbar icon d Or Select Auto de novo from the Tools menu The Auto de novo E and dM feos Demo ee dialogue window x De novo will
140. rbyServer ser verCB SL AC Sample Tasks Running info Properties Selection Det sis SILACS ample mao Total MSI Spectra 3 Total MS MS Spectra Ion Source ESD nano spx ay Fragmentation Mode CID CAD RIFO y and b ions MS Scan Mode FT I1CR Orbtrap MSIMS Scan Mode Unes lon Trap MS Scan Centroid rn MSIMS Scan Centroid fake T 100 100 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000 2100 Jr rh 2X 2Y SILACS ample moo ms 1 RT 0 02 scane2 TICe 3 34E7 1243640 998 5 0 100 200 300 400 500 600 700 800 900 1000 1100 1200 1300 9 1 er 2X 2Y SILACSample mz04 mee mre 1243 64 290 190 03 scan 3 TIC 2 863 116 2 Running Protein Identification 1 In the Project View Frame select SILACSample mzxml Click the data refinement tool a select Data Refine Data Refine from the Tools Data Refine jokes menu Enter the De novo Retention time window For raw Files only min p ar ameter S aS PEAKS Search je aera Da shown below and n TE cli ck OK Correct precursor charges g ys O re Minimum charge 1 Maximum charge Data Refine Merge scans of the same peptide yes no Quantification Filter Options Filter MS MS scans Precursor mass between and and Retention time between Quality value greater than suggest 9 65 Preprocess Options Preprocess MS MS scans O no already done
141. re is also a Peptides List box which displays information about the peptides that matched to the selected protein This list is identical to the Peptide View panel so see this section for more details Peptides List Sequence PEARSE RSD Mricalc Delta M Error File gt Peptides Spectrum 1 DAPOTHY Y AVAVVE 99 0 1603 7993 0 0061 3 8056 OrbiSam Spectrum TSDAMINM 3 WMNLK 99 0 13832 6523 0 0132 3 4869 OrbiSam LE AU HSTVFDNLPHPEDR p Spectrum n n 0 0065 3 9455 OrbiSam i 4 Spectrum 4 0 0 0 0107 6 5511 OrbiSam Below the Peptides List you will see the protein sequence with the matching peptide sequences in blue The darker the blue the more confident the match 1s Matched peptides shown in blue SPIDER matches shown in red 1 MRPAVRALLA CAVLGLCLAD PERTVEWCTI STHEANECAS FRENVLEILE al AGPFVOCWVER TSHMDCIEAI SNNEADAVTL DGGLVYEAGL EKPHNLEPWVYA 101 EFHGTEDNPO THYYAVAVVE KDTDFELNEL RGKKSCHTGL GRSAGWNIPM Sequence Comparison Click on the Sequence Comparison tab to open the multiple sequence alignment window A multiple sequence alignment helps to highlight the differences and similarities between homologous proteins and the variants you ve evidenced from your sample In the above list of proteins mark two or more entries by clicking in their checkboxes Click one of the above buttons to generate the multiple sequence alignment in this frame or in your web browser Identified pepti
142. ree It 1s for quantification based on the relative intensities of extracted 10n chromatograms XICs for precursors in multiple data sets aligned using mass and elution time Sample It is for specifying sample name Reporter ion It is for specifying mass of reporter ion Added Mass The modified mass of a residue Residues The residue to be modified Labeling efficiency It 1s for specifying efficiency of chemical reaction Add label It is used to add a label Delete label It 1s used to delete a label 15 2 3D View In order to produce a 3D view you must first select this in your preferences 1 Click on the Preferences toolbar icon e or select Preferences from the Windows menu A Preferences Performance Computer Performance RMI Connections 2 Select General 8 Derby Database J i Performance Single Core Dual Core Quad Core from the panel on the reum left hand side la Search Engine Show 3D View Ion Settings Fast Loading Files 3 Select Performance and Appl check the Show 3D 9 eee 4 Click Apply 108 Chart View Protein View Peptide View Denovo View When a PEAKS Quantification run is complete a 3D View tab can be found in the Peptide View window ID Sequence PEAKS Score RSD Miz Z Mr Calc Delta Mass Error ppm File RT Scan Heavy Light 3 Peptides Spectrum 4 APTPTGAAEMAVPVK 1438 749 20 2775 SILACSample rmz Hit 6 DA
143. roperties window will open Give your sample a name You can now select file s that pertains to this sample using the Add a file for this sample To remove files or clear the list you can use the Remove from list and Clear list buttons respectively You may also leave Project Location CiPeaks Studio 5 0 derby ServeriserverDB Project Folder CiPeaks Studia 5 0 derby Server serverDB Project 1 Notes Description Example Samples from orbitrap on August 10 12 Type and organization of project 5 Basic Project Steps Sample Properties 1 Project Properties Give this sample a name Sample 1 2 Sample Properties Select Files WS uni Mame Size Date Modified Type onidataliain 266 RAW 171 136 KB 03 02 2006 10 05 00 AM _onidataliaint 269 RAW 32 798 KB O3 02 2006 13 18 00 PM Add a File Far this sample Remove File Fram list Clear lisk Notes about this sample Add another sample Remove current sample notes about the sample for reference 36 4 To add another sample or remove the current sample use the Add another sample and Remove current sample buttons respectively 5 Select the Next button once all relevant files are added to each sample 6 The Instrument Details window will open Select the instrument that was used to generate the experimental data From the drop down menu select General for t
144. rotein structure prediction and PatternHunter software for all types of homology search sequence comparison 128 20 PEAKS Software License This is the same agreement presented on installation It is provided here for reference only If we are evaluating a time limited trial version of PEAKS and we wish to update the software to the full version we must purchase PEAKS and obtain a full version registration key 1 License Subject to the terms and conditions of this Agreement Bioinformatics Solutions BSI grants to you Licensee a non exclusive perpetual non transferable personal license to install execute and use one copy of PEAKS Software on one single CPU at any one time Licensee may use the Software for its internal business purposes only 2 Ownership The Software is a proprietary product of BSI and is protected by copyright laws and international copyright treaties as well as other intellectual property laws and treaties BSI shall at all times own all right title and interest in and to the Software including all intellectual property rights therein You shall not remove any copyright notice or other proprietary or restrictive notice or legend contained or included in the Software and you shall reproduce and copy all such information on all copies made hereunder including such copies as may be necessary for archival or backup purposes 3 Restrictions Licensee may not use reproduce transmit modify adapt or translate
145. rovides the local copy uses the location of PEAKS as the default path Click the Apply button to save any changes you have made Sequest Settings Clicking on Search Engine and then Sequest Settings in the menu on the left hand will open the following window amp Preferences To use Sequest Settings Sequest ped Instrument must click Search Engine Sequest Location the General Fe Browse Ss XTandem Settings button to tell A Omssa Settings Default Sequest Parameter File params PEAKS Ei Sequest Settings C Xcalibur params orbiSample params where to Ion Editor find the Sequest Result Output Folder default path C Xcalibur sequest You must also browse your computer to find the location of the Default Sequest Parameter File params as well as the Sequest Result Output Folder Click the Apply button to save any changes you have made Ion Editor Preferences A Preferences z General Clicking on Ion General E Inst E Editor on the menu me s Search Engine on the left hand will gm Show Decimal Places 28 open the following 9 nes window 95 Decimal places Select the number of decimal places you would like to appear in the ion table The default is set to two decimal places Ion Editor Clicking on Ion Settings and then Jon Editor in the menu on the left hand will open the following window Preferences Ion Editor Choose the i
146. rt the spectrum view ion table or a picture bmp gif or jpg format with ions masses PEAKS and peptides marked o Exit To exit from the PEAKS software safely select this icon or press on the keyboard the Control key simultaneously with the letter Q x Data Refine Merge scans of the same peptide remove noise spectra preprocess within each MS MS spectrum and recover peptide charge state The data refinement options dialogue will allow us to choose and to set parameters for each of these refinement tools d De novo Perform auto de novo for a selected data file spectrum or project Press this after selecting one or more data files or spectra in the Peptide Data Frame An auto de novo options dialogue will allow users to set parameters before beginning q PEAKS Search Perform protein identification on a selected data project Press this after selecting one or more data files or spectra in the Peptide Data Frame A protein identification options dialogue will allow users to set parameters Za SPIDER Search Peptide homologue search tool hs PTM Finder A PTM finder search can be performed on any PEAKS Protein ID and allows the user to be able to identify more PTMs in less time 124 E inChorus Search inChorus a meta protein identification tool can be used to compare and validate results calling upon such search engines as Mascot OMSSA PEAKS Sequest and X Tandem as well as SPIDER Click this icon to set a
147. search instead it will insist that the mass of the peptide returned 1s the same as that of the de novo sequence Non gapped Homology Match this search will allow for transpositions and single point mutations but not insertions or deletions Gapped Homology Match this search 1s the most rigorous will find all types of mutations but it is the slowest of the three search modes Block Match this 1s the most rigorous but most resource intensive search mode taking into account all types of mutations and the positional confidence scores A quick version of this 1s used to create the reconstructed peptides and to generate the final scores in each of the previous search modes This is the only search mode that allows you to use variable modifications 64 General Options Amino acid selection Choose if you would like PEAKS to consider Leucine equal to Isoleucine without a penalty in the score as well as whether Lysine should be equal to Glutamine without penalty Mass tolerance Enter the amount of error in Daltons that PEAKS will allow for when determining the peptide sequences Number of peptides to report Choose how many of the best homologous peptides should be displayed after searching PTM Options PTM Options List The PTM Options list tells PEAKS what kind of post translational modifications to include in its analysis To view additional modifications select the Show unimod box If your desired PTM does not appe
148. sees 88 General Prelere nes oiu ost ttd o m EG EC soi sans ERE EQUES D S aA T Se 89 MSCFUMenE PrelerenCes ooo ORT EATER DEDE PEDIR OAAS 91 Scarch Engine PreferenCeS siseste neenon E Due PEE E EE E Ier EI Ya Deere MERE ISO Deb 93 Ion Editor Pere rences eei E epo REPE Evae NEXT IER Eee Regino aa 95 142 PEAKS CoOnnmeuration siue eO ERE ete eque e e te ER eed eve ege ed Uer URS 97 Enzyme Bro ib cli em 97 PENLCORDPUEALUOT sie sce csccecivccssucesscesscasseituacacessdsceseesieesncesccasseateadoceeseccasveteasvecest 98 Database COMM OU aOiiesiecs t r t 99 Instrument Configuratloli siessen aia Daaa 103 Parameter Configuration sessar araa aS 104 15 PEAKS Quantification oeceisee ei aeo o a eue dao queue guo YD ONES ETE ea PEU MH CREDE Persa eure RR EE dus 106 15 1 Setting up PEAKS Q Parameters ce eee ee eee ee e eee ee eee eee eee e e e e ette eese eoous 106 15 2 9D VICW uisttctniieast esses uada potes pres edha cesses tu EEEE esa vietato cessat eed nt leta tea und 108 15 5 TERA QO WalktBrOUPl iouis ceci edes Caen ee ab ebat he nude ee edu se Coen voe adque ehe nude eee a buque us 109 1 Creaung a PEOJeGEm oot Ir vinee io aada n AO OESE aS a voee e o Pe ri beoe 109 2 Running data FrelInelielio ee eee eoa th ba voe ev SEEY TEE Poe EAR E ERE CE Ra Poe eO SEE P LER RES 111 3 Running PRA KS S6aren S ier tos aasre P Tero Pedo EIE RAT iaat 112 4 R nnin Quantilcatioh iiic ie a ER Ee Ee Orb Ir ist nie ta Ee E Is 113 15 4 S
149. splay the protein with its matched peptides in red SPIDER will also display a reconstructed sequence See page 66 for more information FEE fea feed fee _ Spectrum Spectrum3 ANELLLNVK zo og 803 D Spectrum 4 LSHEFALNGNPQNP 140 O57 547 58 D Spectrum 5 HSTVFDNLPNPEDR god T Spectrum 6 VDDYQEC 1 YLAMVPSHAVVAR 23 0 TEEECET Peptide Details Select peptides For display a1 101 151 201 251 lt FWL gt Denova W CarboxymethylC W I SL BINLINVYININLE 6 uud bys ot Q29443 TRFE BOVIN YLAMVPSHA RL E PETTITT Recon AD CarboxymethylCc WR Cor memi gt aaa IT EHEEEAE MM l HomalagVD D Matched peptides shown in blue SPIDER matches shown in red MPPAVPALLA CAVLGLCLAD PERTVEWCTI STHEANECAS SGPFVSCVEE TSHMDCIEAI SNNEADAVTL DGGLVYEAGL EFHGTEDNPO THYYAVAVVE EKDTDFELNEL RGE RESCHIGL AELTYEELPDP QESIORAAAN FFSASCVPCA DOSSFPELCO ACSNHEPYFG YSGAFECLME GAGDVAFVEH STVFDHNHLPNP 1 GDNTRESVDD YOECYLAMVP SHAVVAETVG GE EDVIWELL 3l i lt QE CarboxymethylC Y LAMVP SHA VVA R FEENVLEILE EPNNLEPVVA GESAGWINIPM LCAGEGTDEC EDRENYELLC NHAQEHFGED 4 Load data 4 1 Data Format Before loading data files into PEAKS you must make sure that the data is in an accessible format PEAKS handles data files in the following formats PKL The file format associated with MassLynx software DTA The file format associated with SEQUEST software MGE The file for
150. t click the New Database button Note that you can also set up a new database in the Database Configuration window The configuration window is the only place that that you can delete databases that you have created For more information on setting up new databases see page 99 Taxonomy selection This list displays the taxa you have chosen for your search If the database selected has taxon information available you can click on the Set taxa button Otherwise the whole database will be searched The selections correspond to established hierarchy 1 e selecting Mammalia will search all of horse cow rat mouse human etc Paste FASTA sequences If you already know the sequence of the protein s you are looking for select Paste fasta sequences and paste the sequence in the space provided in fasta format Alternatively if you want search the same sequence regularly it is recommended to simply create a small text file and configure it as a database for PEAKS Advanced Options PEAKS needs to have some de novo sequences before database searching since PEAKS uses sequence tags to perform database searching As such the option of doing de novo prior to protein ID is presented here In most cases the same values for instrument error enzyme and PTM can be used in de novo and in protein ID but you have the option of using one of your saved de novo parameter sets for the de novo portion Select one from the drop down l
151. t these last two steps In our case we are not going to add any more samples so we can just click Next Add a File For this sample Remove file From list Clear list Sample Notes Description Add another sample Remove current sample 115 amp amp New Project The following Steps Instrument Details section will tell EEE PEAKS which type of mass 1 Project Properties Instruments s used to acquire the data 2 Sample Properties T Instrument petal Instrument Details LTO FT Ultra Hybrid FT Trap spectrometer Was used to generate 4 LTQ FT Ultra Hybrid FT F Ion Source ESI nana spray the data This MS Scan FT ICR Orbitrap LTQ FT Ultra Hybrid FT F Fragmentation Type CID CAD IRMPD ty and b ions sample Was Fj LTQ FT Ultra Hybrid FT F Msn Scan Linear Ion Trap derived from a i i E Default Parent Mass Tolerance 0 01 Da Thermo LT Ego laa eem Ped Default Fragment Mass Tolerance 0 5 Da Orbi tra id k LTQ FT Ultra Hybrid FT T MS Data Centroided Q yes p Misc ERU ORE TER MS MS Data Centroided C yes the box beside LTQ FT Ultra LTG FT Ultra Hybrid FT Tr Hybri d FT Tr ap LTO FT Ultra Hybrid Trap to select this lt li gt instrument Upon clicking Next a sample project will be created Your Main Processing Screen should look something like this PEAKS a ee Fie Tools Window Help GBRBUBI amp 4A OQ o V Project View ShACSpeple mdM x A O Ide
152. t Iu Ee a enini cccisacatendontisccbieiedeotsedeadineisiatenes 76 I2 C omiplex A TAN YSIS 5e eeco s eeu YE eo ASPIRE SEES EY PRSE RR CER SEN RE ESSEN d EUR NE Er EE NNUS PR QUE RE a PEU 78 12 1 Creating a project for complex system eee e eee eee e e e e eee eee ette eee ees essone 78 12 2 MCCS EATING data ATIANY sls uio oec ek e eS Ee YE eS Fe UE ao Ee ee S Pei YES te teo is deci 79 13 Exporting Data Reports and Printing e ecce e eee eee eee eee ee e eese 81 13 1 Export Data un mzxml OF ig isse teer tasa eua eet n eee eaa na HEURES eel Ys a eaa Eo eoe sa Peau e gno a 81 13 2 Export Peptide Results in PepXML Format eee e eee ee eee e e eee eene 82 13 3 Export Results in Excel Format cccsssssssssccccsssssssssssccccscccssssssssccccssssssees 82 13 4 Print Tables and Graphs for Publication ccce ecce eee e e eene 84 De novo Imase Fes 2 eoo eere eau a re eto aquo os teo bea Vo oan toe Ee eva d quete eo E een eae 84 Protein ID Tima Ge Filis eR 85 int horus Pima Ge PINGS vies csscasescsscusccccaseuetesesebeateacesesevesenseccancaasesusshenceanscsswsusepesceane 86 Compare Image Pies io Ert pO Evi PSU EDI taU Eie e MEE 86 14 Advanced Configuration and Environment Preferences 88 14 1 PEAKS Environment Preferences sssccsscccsssssssssscssccccccccssssssssccccsssss
153. t when PEAKS reads our database Select the database that 1s similar to yours from the dropdown list to fill the textboxes with the appropriate parsing rules A note on parsing rules Apart from starting with a greater than symbol the precise syntax of the FASTA title line varies from database to database For this reason PEAKS uses Java Regular Expressions to define how the accession string and the description text should be parsed from the FASTA title line A note on using a delimiter Some databases use one entry to represent multiple protein entries The FASTA headers are concatenated with a delimiter Since some of these databases use unprintable control codes as delimiters PEAKS will use the equivalent ASCII decimal code to represent them For example the NCBI NR database uses CTRL A as a delimiter so the user should input 1 as its equivalent decimal delimiter as listed here 10 To be able to do PEAKS Protein ID using a specific taxonomy you will need to download some files and place tell PEAKS where to find them in the Taxonomy Options panel 11 To download the taxonid file click the Download button The following window will appear Download options You have choose to download the database From Ftp Ftp ncbi nih gov pub taxonomy gi_taxid_prot dmp gz Click YES Eo invoke your default FTP client software and download automaticlly Tt may Fails due to the setting conflict of default FTP client software Click
154. task to have inChorus perform a user s analysis easily Q Quantification Where users require abundance ratio results PEAKS Q provides insight Click here to analyze results from ICAT 1TRAQ Label Free SILAC N terminal and User Defined Labeling techniques Configuration Set up enzymes post translational modifications databases instruments and parameters oe b e e e e e e e e Preferences Organize general properties such as directories search engines and ion editing capabilities p Mass Calculator The Mass Calculator is a simple tool to help us determine the molecular weight of a peptide Clicking this icon will make the mass calculator appear Project View The project view allows optimal organization and greater control when managing multiple files at once As different projects may be active at once it is important to understand the different categories and levels presented by PEAKS FX Tree Root HH Project Node Y Filter Result Sample Node O File Node Combine Results ir Compare Results Search Engines Within the Project View these icons are used to represent which method search engine a particular file was interpreted by Mi Mascot M OMSSA 8 Sequest X X Tandem Main Processing Window Toolbar ill Profile Mode uli Peak Mode lil Return to original size 125 Zoom X axis ai Zoom Y axis 18 3 Mass Calculator Click the Mass Calculator icon on the toolbar gt Or choose Mass C
155. techniques Results generated from PEAKS Q have high accuracy and can be performed over a wide dynamic range Please note that the label free quantification protocol is not included in this first release PEAKS 5 uses project based data management which allows users to process simultaneous runs and easily compare contrast samples within one project 1 3 Workflow Data loading Data refine Identification Quantification 3 PEAKS DB processing searching quantification Sequencing merging searching MS MS charge SPIDER quantification quality PTMFinder Label free filter quantification Exporting Data Analysis Publication exporting reporting 1 4 Guidelines for Using this Manual This user s manual is intended to help you get started with PEAKS 5 It will describe its functionalities show how to customize PEAKS to your applications provide a task based reference and troubleshooting We recommend reading the walkthrough in Chapter 3 using the sample data provided 1 5 Scope PEAKS users are assumed to be familiar with computer usage and the operating system environment As such it is beyond the scope of this manual to instruct the user on the use of windows dialogue boxes menus file storage etc Please refer to the operating system s manual or computer help books for such information Similarly PEAKS users are expected to be familiar with mass spectrometry standard operating practices and data 1 6 Service and Support In add
156. teen eee etes stesse ees esese eese etenoe 129 1 Introduction to PEAKS 5 1 1 Main Features PEAKS is an innovative software system designed to derive amino acid sequences and identify proteins using tandem mass spectrometry data from all major mass spectrometry vendors PEAKS incorporates de novo sequencing results into the database searching process for peptide protein identification It does this by generating sequence tags which are used in conjunction with fragment ion mass matching to speed up the search remove false positive matches and find peptides with interesting sequence variations or modifications that would prevent them from being otherwise identified Our meta protein search tool inChorus allows users to use multiple search engines PEAKS Sequest Mascot X Tandem and OMSSA to expand sequence coverage and increase confidence Another tool SPIDER is used to reconstruct the correct sequence using the de novo sequence and a homologous peptide 1 2 New Features in PEAKS 5 We have many new features in PEAKS 5 which will be explained throughout this manual PEAKS 5 is now capable of handling very large data sets Our protein identification 1s more sensitive and generates less false positives PEAKS 5 also has improved identification of PTMs with our new PTM finder BST makers of PEAKS has also created a quantification module which will allow users to automatically quantify proteins from experiments using both label and label free
157. tein ID Moving Updating a Database If you choose to move a database to another directory or delete it entirely you need to notify PEAKS You must remove the database from the list and re load it Until you do so the database name will appear in red in the list of databases and any protein identification using that database will fail If you choose to update the database perhaps by downloading the latest database file and overwriting the old database file PEAKS will show the database information in light gray A light grey colour could also mean that the database does not have header information Best practices configuring databases for use with X Tandem At the time of this writing X Tandem had trouble searching through large databases and would crash It is therefore suggested that X Tandem only be used with small databases or if used with a large database a taxon should be specified The NCBInr and SwissProt databases are ideal for this purpose Best practices configuring databases for use with OMSSA At the time of this writing we could not use OMSSA with databases that were not in NCBI format or SwissProt format and have those results available to inChorus 102 Also a bug in OMSSA prevents us from easily using databases with OMSSA when they are stored in a folder that contains a space in its path This creates problems when PEAKS creates temporary databases on our behalf To avoid this best practices suggest that you
158. ters boxes will be populated 5 Press the OK button to initiate de novo sequencing 6 2 De novo Sequencing Results Once de novo sequencing is finished the following window will open E Sequence TUG ALC Rank mjz Z Mass File RT Scans 2 1 DNPQTHYYAVAVVK 9 43 0 67 1 802 91 2 1603 7993 OrbiSample mzxML 0 07 2 2j 1 DNPKTHYYAVAVVK 9 43 0 67 2 802 91 2 1603 8357 OrbiSample mzXML 0 072 LA 1IDNPAGTHYYAVAVVK 9 11 0 61 3 802 91 2 1603 7993 OrbiSample mzXML 0 072 1IDNPGATHYYAVAVVK 9 11 0 61 4 802 91 2 1603 7993 OrbiSample mzXML 0 072 A 1 DNPQTHYYGLAVWK 8 29 0 59 5 802 91 2 1603 7993 OrbiSample mzXML 0 072 2ITSDANLDWNNLK 6 97 0 58 1l 695 84 2 1389 6523 OrbiSample mzxML 0 314 2 MGDANLOWNNLK 6 14 0 51 2 695 84 2 1389 6348 OrbiSample mzXML 0 314 2 STDANLDWNNLK 6 14 0 51 3 695 84 2 1389 6523 OrbiSample m2XML 0 3114 2IGMDANLDWNNLK 6 14 0 51 4 695 84 2 1389 6348 OrbiSample mzXML 0 314 2 TSDANLDWMPLK 6 26 0 52 5 695 84 2 1389 6599 OrbiSample mzXML 0 3114 v Immonium b b H20 a C Seq y y H20 zZ 2 y 2 1 88 04 116 03 98 02 88 04 133 06 D 14 a 2 87 06 230 08 212 07 202 08 247 10 N 1489 76 1471 77 1472 74 1473 75 74539 B 3 70 07 32717 30915 29914 34416 P 1375 71 135771 135871 1359 66 688 37 12 4 101 07 455 19 437 18 427 19 4
159. the Software in whole or in part to others except as otherwise permitted by this Agreement Licensee may not reverse engineer decompile disassemble or create derivative works based on the Software Licensee may not use the Software in any manner whatsoever with the result that access to the Software may be obtained through the Internet including without limitation any web page Licensee may not rent lease license transfer assign sell or otherwise provide access to the Software in whole or in part on a temporary or permanent basis except as otherwise permitted by this Agreement Licensee may not alter remove or cover proprietary notices in or on the Licensed Software or storage media or use the Licensed Software in any unlawful manner whatsoever 4 Limitation of Warranty THE LICENSED SOFTWARE IS PROVIDED AS IS WITHOUT ANY WARRANTIES OR CONDITIONS OF ANY KIND INCLUDING BUT NOT LIMITED TO WARRANTIES OR CONDITIONS OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE LICENSEE ASSUMES THE ENTIRE RISK AS TO THE RESULTS AND PERFORMANCE OF THE LICENSED SOFTWARE 5 Limitation of Liability INNO EVENT WILL LICENSOR OR ITS SUPPLIERS BE LIABLE TO LICENSEE FOR ANY INDIRECT INCIDENTAL SPECIAL OR CONSEQUENTIAL DAMAGES WHATSOEVER EVEN IF THE LICENSOR OR ITS SUPPLIERS HAVE BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGE OR CLAIM OR IT IS FORESEEABLE LICENSOR S MAXIMUM AGGREGATE LIABILITY TO LICENSEE SHALL NOT EXCEED THE AMOUNT PAID BY LIC
160. the menu on the left hand side will open the following window amp Preferences x General Bruker yep baf fid files Instrument E ABI wif B Bruker yep baf Fid a Shimadzu AXIMA ru as E Varian xms Search Engine Default compass File location C Program Files Common Files Bruker Daltonik AIDA export CompassXport exe 3i Ion Editor Raw file convertor options Bruker Fid File may contain several files da you want to merge them into one data set C yes no Default compass file location Click Browse to tell PEAKS the location of the Compass Xport file converter Raw file converter options Bruker fid files may contain several samples By default these samples are not merged into one data set Select yes if you would like PEAKS to merge all the samples into one data set Click the Apply button to save any changes you have made Shimadzu AXIMA run Clicking on Instrument and then Shimadzu AXIMA run in the menu on the left hand side will open the following window 02 A Preferences a General Shimadzu AXIMA run Instrument i ABIL wiff Shimadzu runzxml exe File location E Bruker yepfbaf Ri ef Shimadzu AXIMA ru s gt Variant xms Search Engine Ion Editor Click Browse to tell PEAKS the location of the Shimadzu run2xml exe file Click the Apply button to save any changes you have made Varian xms Clicking on Ins
161. tification calculated from the confidence on the ten best peptide hits for this protein and normalized against the other identified proteins Coverage The number of amino acids in the protein sequence that have been explained by the identified peptides Expressed as a percentage of the total length of the protein matching to a peptide from this protein Marked A multi function checkbox By default unchecked but we can use this to select proteins for export or multiple sequence alignment Description The part of the protein s header information as parsed from the database usually it contains the name of the protein The columns themselves can be customized Right click anywhere in the report and choose Toggle Column from the pop up menu The sub menu that appears shows a checkmark in each of the columns that are currently showing Click any of them to show or hide a column These settings will apply to all your reports 59 Sequence Browser The Sequence Browser tab is selected by default Clicking on a protein in the index will display the sequence of that protein below in the Sequence Browser panel Clicking on the hyperlink of the accession number of the protein shown in blue will open a new window containing the webpage of the database that you searched for protein ID page in a new window NCBI BLAST search of p Q29443 TRFE BOVI Link to retrieve entries containing this sequence From NCBI Entrez The
162. tion will help PEAKS decide which internal parameters for weighing fragments and amount of noise to use during PEAKS auto de novo sequencing and PEAKS protein ID database search Select LIT FT if alternating hi res low res modes are used allowing the algorithm to determine the mass analyzer from the scan header You can also use the Advanced Options to specify additional parameters Precursor Mass Search Type Select Monoisotopic or Average For ion trap instruments it is usually beneficial to allow the PEAKS protein ID database search to use an average mass Parent and Fragment error mass tolerance User specified values These will appear on the PEAKS de novo and PEAKS protein ID options screens when the instrument is selected Target Ions Select which ions that you would like PEAKS de novo and Protein ID to focus their search on You must click the Add Update button for the changes to be saved Your new instrument will now appear in the Instrument List where you can access it later If you wish to delete an instrument that you created select the appropriate instrument and click the Delete Instrument button Parameter Configuration From the Configuration window select Parameters from the left hand to change your parameter configurations Please note that you can only view and delete parameters from within this parameter window From the Parameter type drop down list at the top of the screen you
163. topic PO Residues that can be modified Po Formula PO Rule PO Fill in the following information Name this name will appear in the PTM list for future use after it is saved Monoisotopic mass the mass that the residue gains or loses as a result of the PTM Enter this value numerically Neutral loss mass the mass that the modified residue loses as a result of fragmentation Ex 28 would signify a loss of 28 Daltons This is optional Chemical formula the chemical formula of the PTM This should correspond to the mass listed above This is optional Residues that can be modified Enter residues that can be modified anywhere residues that can only be modified if they are at the N or C terminus or in the middle only Rule Enter comments for reference This is optional 53 Please note that you can also configure your PTMs in the Configuration panel See page 98 for more information Max variable PTM per peptide To reduce uncertainty limit PEAKS de novo sequencing vocabulary by restricting the number of variable PTM found on a peptide Specify a number by typing it into the box To lift such restrictions type a very large number longer than the length of the peptide Database Options Database to search Select from this dropdown list one of the FASTA databases configured in PEAKS To edit an already existing database click on the Edit Database button If the desired database is not in this lis
164. trument and then Varian xms in the menu on the left hand side will open the following window Preferences General Varian XMS Instrument Default xmlrai exe location H E ABIC wifF E ker veplbaf na EEEEREEEEREIIIEEEEEETECLILLLOLLUL ECCINUSPVOCSZCO LLINUL AaoUL4 S5 Shimadzu AXIMA ru Varian xms Search Engine Ion Editor Click Browse to tell PEAKS the location of the xmlrai exe file Click the Apply button to save any changes you have made Search Engine Preferences Preferences General Mascot Settings Instrument oe ee S seachtnone Host name or IP aces Clicking on Search E Mascot Settings Engine and then Mascot E XTandem Settings Settings on the menuon orse senros vu rectory i l a User name Wa the left hand side will open ai User name leu Ion Editor the following window password o Save Password 93 In this window you will tell PEAKS how to access your Mascot server if applicable Enter the Host name or an IP address Port Virtual Directory as well as your user name password and email address To make sure that you entered everything correctly and that the server is working click the Test Connection button If you would like to save your password so that you don t have to enter it every time check the Save Password box Click the Apply button to save any changes you have made X Tande
165. um charge to start using multiply charged products 3 Fraction of product peaks below precursor to determine 1 precursor 0 95 Peak intensity cutoff Number of top intensity peaks in first pass lo fraction of most intense 6 T Ions to search 1 b gt Ions to search 2 v Click the Ok button When the inChorus search is complete you should see the following new additions in the Project View panel 4 PEAKS 1 21 Jan 09 11 38 XY S TANDEM 2 21 Jan 09 11 39 OMSSA 3 21 Jan 09 11 40 e INCHORLIS 4 21 Jan 09 11 40 Presented here are individual reports for PEAKS X Tandem and OMSSA as well as an inChorus report that compares the individual reports To see each of these reports click on the report that you would like to see in the Project View panel 27 The Peptide View results for the PEAKS Protein ID search can be seen below ID Sequence PEAKS Score 95 RSD Miz Z Mr Calc Delta Mass Error p File RT Scan 43 Peptides Spectrum 1 DNPOTHYYAVAVVK 0 0061 3 8056 OrbiSample mz Spectrum 2 TSDANIN 3 WNNLK 0 0132 9 4869 OrbiSample mz 0 0062 6 0879 OrbiSample mz 6 0879 OrbiSample mz Spectrum 3 ANELLINVK Spectrum 3 ANEILINVK HSTVFDNLPNPEDR 3 9455 OrbiSample mz Spectrum 5 0 0107 6 5511 OrbiSample mz La Spectrum 4
166. urvey Alignment Error Map 4 Running Quantification Select the PEAKS Search result file and click the PEAKS Quantification toolbar icon or selecting Quantification from the Tools menu The quantification parameters window will open Enter the parameters as shown below and click OK 113 0 042 Scan 11 11 3 e 13 le 11 10 13 EUM NEUEN 10 3 8 m 05 HEN a 4 Quantification Once completed the Quantification pr otein m quantification result Daka Refine Mass Error Tolerance 0 Upper Bound of Precursor Charge 4 will be di splayed in Dennis Retention Time Range the same PEAKS PEAKS Search Label Options Protein ID result SPIDER Search Labelling occurs at the MS MS level eg iTRAQ window th at you Labelling occurs at the MS level eg ICAT PTM Finder Mon selected earlier Y Quantification Sample Reporter Ion Da Labelling Efficiency The results are E IIS listed as a Ratio of 117 115 114 112 and as Standard Deviation of 117 115 114 112 They are highlighted in the red box below For example the relative protein ratio for the top ranked protein is 4 73 with a standard derivation of 0 38 Accession ID Mass PEAKS Scor Ratio 117 115 114 112 SD 117 115 114 112 Display Coverag Query mat Marked Descri DB Search F o P00722 BGAL_ECOLI 1 116482 8 96 59 4 88 0 34 4
167. use this function PEAKS must have access to a protein or EST database in FASTA format or an EST database of DNA sequences You can point PEAKS to an existing database on your system or download one Additionally you can associate taxonomy with certain databases WARNING Downloading a database can take a long time 8 hours depending on connection speed Most only take 20 30 minutes From the Configuration window select Database from the left hand to change your database configuration The Database list at the top of the screen will show you databases that you have already configured Select one of these files to see the details in the Database Details panel below Configure a new database 1 Select the New Database button on the right hand side of the Database List You will now be filling in the specifics for your database in the Database Details panel below Database Details FASTA Format database MCEI nr V Basic Options Database name MCEI nr Download Database Path CAPEAKS databaselNCBI nr EST database Advanced Options Faska Title Farmat Rule bo parse accession id From FASTA title Vg tio Rule to parse description From FASTA title irm AccessionJid URL http www ncbi nim nih govfentrez viewer Fogi db protein amp yal lt Accession ID gt Po Delimiter e Taxonomy Options Laxonid CHPEAKS databaselNCBInrlgi taxid prot dmp gg 000 database NCBI nrigi
168. ve protein per group All Protein and Peptide Results Options d Export Data Properties Export Search Parameters _ Export Filter Conditions Select the Export Destination If you would like to export all of the protein and peptide results select the All Protein and Peptide Result s otherwise select one of the other options where you can limit which results are exported Select the appropriate boxes if you would like to export Data Properties Search Parameters and Filter Conditions to Excel Finally you need to select the Export Destination by clicking the Browse button Then click OK If you export De novo results to Excel the de novo sequencing results will be exported and you have the choice to also export Data Properties Search Parameters and Filter Conditions See below Export Excel Result Report Select the Type of Results to Export Export Search Parameters Export Filter Conditions Select the Export Destination Excel File 83 13 4 Print Tables and Graphs for Publication In order to export an image file right click on the results file that contains the appropriate image file and select Export Statistics Graph y Project View EE Mew Project 3 Jl Sample 1 a OrbiSsample mzXML Er DENOVO 1 19 Jan 09 10 43 i haf PEAKS 22 19 Jan 09 15 31 NI STANDEM 23 19 Jan 09 15 32 QMS5A 24 19 Jan 09 15 34
169. would signify a loss of 28 Daltons Chemical formula the chemical formula of the PTM This should correspond to the mass listed above Residues that can be modified Enter residues that can be modified anywhere residues that can only be modified if they are at the N or C terminus or in the middle only Rule you can enter a comment for your reference You must click the Xe ec P Enzyme PTM List inde aus SUNON M Med on Methionine for the changes to be Database lt Built In gt 4 hydroxynonenal HNE J ___deeter uilt In gt Acetylation K saved Your new Instrument leo a ata AMA I Parameters lt Built In gt Amidation PTM will now appear ae Cee ee nerd Nu in the PTM List Cc where you can access buen ee P PTM name Oxidation on Methionine 1t later i If you wish to Mass Monoisotopic 15 994915 delete A PTM that you Neutral loss mass Monoisotopic 0 0 e Residues that can be modified M Anywhere v created select the RU lo appropriate PTM and Rule click the Delete PTM button 98 The example listed below is one where we knew that only methionine was oxidized Note For information on defining new PTMs on the fly for PEAKS de novo or PEAKS Protein ID see pg 45 or pg 53 respectively Database Configuration In addition to de novo sequencing of peptides PEAKS 5 also has the ability to search through a database search to identify proteins In order to
170. y selecting a sample the E Pu Sample operation applies to all files in the sample It means all 7 fy DENOVO 51 19 Jan 09 13 31 spectra from different files are processed in a single i PEAKS 11 19 Jan 09 14 36 run The result node is at same level as selected data 29 ETD 01 RAW node Pei DENOVO 1 19 Jan 09 12 52 0 pf PEAKS 1 19 Jan 09 13 17 PEAKS 1 is the PEAKS Protein ID result for file eG ETD 02 RAW ETD OLRAW whereas PEAKS 11 is the PEAKS C FTD_03 RAW Protein ID result for all three fractions of the ETD 1 cr sample Note that the result of a sample may not be the sum of the results of all files in the sample 12 3 Comparing results PEAKS 5 provides a Compare Results function to align differentiate two or more results To use the Compare Results function hold down the Ctrl key and select two or more result files that you would like to compare Click on the right mouse button and select Compare Results FY Project view Below you will see a comparison of the i g ME m PEAKS protein ID results PEAKS 11 Da PEAKS 22 and PEAKS 32 generated for the 0 roD DERE 9 Jan 09 14 36 i BG ETD ot RAW three samples mentioned in the previous be DEMOVO 1 19 Jan 09 12 52 ER LE PEAKS 1 19 Jan 09 13 17 section ETD CID and CID ETD ETD nz RAW 9 ETD 3 RAW GJ CID o rg DENOVO 101 19 Jan 09 15 09 ofa PEAKS 21 19 Jan 09 17 11 ZIEA EAKS 22 19 Jan 09 17 34 E cID ni RAW
171. you would like to use from the dropdown list The inChorus search will be performed on all species in the database unless specified by the user If this database does not appear in this list refer to page 99 to configure your databases To specify which taxa you would like to search click on the Set Taxa button You will need to use the Ctrl key to make multiple selections To import your file click the Browse button that is found beside the appropriate search engine Find the file that you would like to import and click Open Once you have selected the file s that you would like to import and have selected the options for any other search engine searches you would like to perform click OK 71 10 2 inChorus Results View When the inChorus search is complete the Project View panel should contain a separate results file for each search engine that you selected as well as an inChorus report that combines the results from the multiple search engines See an example below p PEAKS 3 12 Jan 09 14 25 MT XTANDEM 4 12 Jan 09 14 26 ye S54 5 12 Jan 09 14 23 2 INCHORUS 6 12 Jan 09 14 28 De novo Peptide and Protein Views Each results file for the 3 party search engines looks very similar to the PEAKS protein ID results file page 55 with a few small differences Firstly there is no De novo View or Chart View and secondly the scoring will be specific to that search engine For example the score f

PEAKS Studio Manual 5.0 - Bioinformatics Solutions Inc.

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